A nanoparticle material capable of inducing cell immune death, and its preparation method and application

Abstract

The present invention provides a nanoparticle material that can induce cell immune death, as well as its preparation method and application, which belong to the field of biomedicine technology. The nanoparticle material provided by the present invention includes a functional protein core and a polymer layer grown in situ on the surface of the functional protein core, and the polymerization monomer of the polymer layer includes an endoplasmic reticulum targeting ligand and a positively charged functional group compound. The present invention grows a layer of polymer network structure containing positively charged functional groups and endoplasmic reticulum targeting ligands in situ on the surface of the functional protein, and the resulting nanoparticle material can be efficiently internalized into cells, escape from lysosomes, be targeted to the endoplasmic reticulum, and overcome reverse transduction to be successfully retained in the endoplasmic reticulum, ultimately generating endoplasmic reticulum pressure and triggering a series of immunological signals. In addition, the functional protein core can also synergize with the polymer network structure to achieve efficient and synergistic anti-tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanoparticle material capable of inducing cell immune death, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer immunotherapy has transformed the way cancer is treated by mobilizing the host immune system to recognize and destroy cancer cells.

[0003] Currently, most cancer immunotherapies are designed based on the cancer-immunity cycle, a self-propagating process that can elicit potent antitumor immune responses. However, low immune cell infiltration and immunosuppressive networks within the tumor microenvironment (TME) diminish tumor immunogenicity and severely suppress host immune responses. Recent studies have demonstrated that tumor cells undergoing immunogenic cell death (ICD) exert vaccine-like functions to generate antitumor immunity, thereby converting "cold" tumors into immunogenic "hot" tumors. ICD is considered a stress-induced process in which endoplasmic reticulum (ER) stress is required to release tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs) from tumor cells, providing antigenicity and adjuvant properties, respectively. Recently, several clinical trials have demonstrated that pretreatment with ICD-inducing agents significantly improves response rates and survival to checkpoint blockade-based immunotherapies. To date, numerous ICD-inducing strategies have been developed, including treatment with chemotherapeutic agents such as anthracyclines and chemopreventive PP1 / GADD34 inhibitors, as well as physical induction strategies such as photodynamic therapy and radiotherapy. Recently, some highly efficient nanoparticle-based ICD inducers have been developed. Currently, all nanoparticle-based ICD inducers are chemical inducers or photosensitizers delivered by nanoscale drug carriers. In these strategies, ICD is induced by the payload, while the nanomaterials have no immunomodulatory function. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a nanoparticle material that can induce cell immune death, and a preparation method and application thereof. The nanoparticle material provided by the present invention has good efficacy in inducing cell immune death.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a nanoparticle material capable of inducing cell immune death, comprising a functional protein core and a polymer layer in situ grown on the surface of the functional protein core, wherein the polymer monomers of the polymer layer include acrylamide, an endoplasmic reticulum targeting ligand, and a positively charged functional group compound;

[0007] The structural formula of the endoplasmic reticulum targeting ligand is shown in formula (1a) or formula (1b); the structural formula of the positively charged functional group compound is shown in any one of formulas (2) to (7);

[0008]

[0009]

[0010] Preferably, the functional protein core is one or more of serum albumin, urate oxidase, catalase, glucose oxidase, horseradish peroxidase, superoxide dismutase, lactate dehydrogenase, acetaldehyde dehydrogenase, ethanol oxidase and acetaldehyde oxidase.

[0011] Preferably, the particle size of the nanoparticle material capable of inducing cell immune death is 10 to 50 nm.

[0012] The present invention provides a method for preparing the above-mentioned nanoparticle material capable of inducing cell immune death, comprising the following steps:

[0013] The buffer solution of the functional protein is mixed with N-acryloyloxysuccinimide to carry out a grafting reaction to obtain a double-bond functionalized protein;

[0014] The buffer solution of the double-bond functionalized protein is mixed with acrylamide, an endoplasmic reticulum targeting ligand, a positively charged functional group compound, a cross-linking agent and an initiator to carry out a polymerization reaction to obtain a nanoparticle material capable of inducing cell immune death.

[0015] Preferably, the method for preparing the endoplasmic reticulum targeting ligand comprises the following steps:

[0016] Under the action of an acid-binding agent, a compound having a structure shown in formula a undergoes a condensation reaction with (meth)acryloyl chloride to obtain an endoplasmic reticulum targeting ligand;

[0017]

[0018] Preferably, the molar ratio of the N-acryloyloxysuccinimide to the functional protein is 20 to 100:1;

[0019] The grafting reaction temperature is 4 to 37° C., and the time is 1 to 24 hours.

[0020] Preferably, the cross-linking agent is N,N'-methylenebisacrylamide;

[0021] The initiators are ammonium persulfate and tetramethylethylenediamine.

[0022] Preferably, the molar ratio of the functional protein to acrylamide, endoplasmic reticulum targeting ligand, and positively charged functional group compound is 1:(2000-5000):(150-250):(200-1000).

[0023] Preferably, the polymerization reaction temperature is 4 to 37° C., and the time is 1 to 24 hours.

[0024] The present invention provides the use of the nanoparticle material capable of inducing cell immune death in the preparation of anti-tumor drugs.

[0025] The present invention provides a nanoparticle material (abbreviated as NanoICD) that can induce cell immune death, including a functional protein core and a polymer layer grown in situ on the surface of the functional protein core, wherein the polymer monomers of the polymer layer include an endoplasmic reticulum targeting ligand (abbreviated as ETL) and a positively charged functional group compound (abbreviated as PG). The present invention creatively constructs a new type of nanomaterial that can target the endoplasmic reticulum and be retained in the endoplasmic reticulum by simulating the process of excessive accumulation of unfolded or misfolded proteins in the endoplasmic reticulum in cells to generate endoplasmic reticulum pressure. The present invention in situ grows a layer of polymer network structure containing positively charged functional groups and endoplasmic reticulum targeting ligands on the surface of functional proteins. NanoICD can be efficiently internalized into cells, escape from lysosomes, target the endoplasmic reticulum, and overcome reverse transduction to be successfully retained in the endoplasmic reticulum, ultimately generating endoplasmic reticulum pressure and triggering a series of immunological signals. The functional protein core can also synergize with the polymer network structure to achieve efficient and synergistic anti-tumor immunotherapy. This invention overcomes the limitation of traditional nanomaterials that are only used as nanocarriers in tumor immunity by rationally designing the surface of nanoparticle materials. It can directly regulate the immune system without the help of conventional immunomodulatory drugs, providing new insights into the design of advanced nanomedicines for cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preparation process and functional diagram of nanoparticle materials that can induce cell immune death;

[0027] Figure 2 The particle size and TEM image of NanoICD;

[0028] Figure 3 The results of the determination of ETL content on the NanoICD surface are shown in Figure 2, where a is the UV spectrum of ETL; b is the standard curve of ETL concentration and UV absorbance; c is the number of ETL on the NanoICD surface under different feed ratios;

[0029] Figure 4 The flow cytometry results of NanoICD-induced cell CRT externalization;

[0030] Figure 5 The results of the intracellular distribution of NanoICD are shown in Figure 2, where a is the distribution of NanoICD in cells; b is the colocalization analysis result;

[0031] Figure 6 The binding force between NanoICD and endoplasmic reticulum was measured by QCM.

[0032] Figure 7 The results of the NanoICD-induced HMGB-1 efflux assay are shown in Figure 5, where a represents the efflux of HMGB-1 from the cell nucleus after NanoICD treatment; b represents the HMGB-1 content in the cell supernatant after NanoICD treatment.

[0033] Figure 8 The results of the NanoICD / BSA-induced EIF2a phosphorylation experiment, where a is the flow cytometry result of the EIF2a phosphorylation level after NanoICD treatment; b is the WB result of the EIF2a phosphorylation level after NanoICD treatment;

[0034] Figure 9 Figure 5 is the RNAseq analysis result, where a is the heat map and hierarchical cluster analysis of differentially expressed genes in B16F10 cells after NanoICD / BSA treatment; b is the top 30 enriched pathways in GO analysis of B16F10 cells treated with NanoICD / BSA; c is the gene enrichment analysis (GSEA) result of B16F10 cells treated with NanoICD / BSA;

[0035] Figure 10 Figure 5 is the test result of NanoICD / BSA activating the anti-tumor immune system in vitro, where a is the flow cytometry result and statistical results of NanoICD / BSA inducing dendritic cell maturation; b is the flow cytometry result and statistical results of NanoICD / BSA promoting dendritic cell maturation and phagocytosis of tumor cells; c is the flow cytometry result and statistical results of NanoICD / BSA promoting dendritic cell antigen presentation;

[0036] Figure 11 The results of the NanoICD / BSA tumor growth inhibition test are shown in Figure 1, where a is a schematic diagram of the NanoICD / BSA tumor inhibition dosing window; b is the tumor growth curve after NanoICD / BSA treatment;

[0037] Figure 12 The flow cytometry results of NanoICD / BSA inducing CRT eversion in vivo;

[0038] Figure 13The results of NanoICD / BSA activating ICD-related anti-tumor immunity in vivo, where a is the result of NanoICD / BSA inducing DCs maturation in vivo; b is the result of NanoICD / BSA generating memory T cells in vivo;

[0039] Figure 14 These are the test results of NanoICD / BSA promoting T cell tumor infiltration, T cell proliferation, and T cell tumor killing, where a represents the result of NanoICD / BSA promoting T cell tumor infiltration in vivo; b represents the result of NanoICD / BSA maintaining T cell proliferation in vivo; and c represents the result of NanoICD / BSA promoting T cell tumor killing in vivo.

[0040] Figure 15 This is the result of NanoICD / BSA inhibiting tumor recurrence;

[0041] Figure 16 The results show that NanoICD / BSA inhibits tumor lung metastasis;

[0042] Figure 17 is the biodistribution of NanoICD / CAT-PCA in vivo;

[0043] Figure 18 The figures are the results of the in vivo anti-tumor growth test of NanoICD / CAT-PCA, where a is the inhibition of tumor growth by NanoICD / CAT-PCA; b is the survival period of tumor-bearing mice after NanoICD / CAT-PCA treatment; and c is the weight change of tumor-bearing mice after NanoICD / CAT-PCA treatment.

[0044] Figure 19 The results of NanoICD / CAT-PCA promoting T cell infiltration are shown in Figure 1, where a is the T cell infiltration in the tumor after NanoICD / CAT-PCA treatment; b is the statistical result of T cell infiltration in the tumor after NanoICD / CAT-PCA treatment; c is the immunofluorescence staining of T cell infiltration in the tumor after NanoICD / CAT-PCA treatment;

[0045] Figure 20 Figure 5 shows the results of NanoICD / CAT-PCA reshaping the immunosuppressive tumor microenvironment, where a&b are the flow cytometric analysis and corresponding quantitative analysis of M2 tumor-associated macrophages in the tumor after NanoICD / CAT-PCA treatment; c&d are the infiltration of Tregs cells in the tumor after NanoICD / CAT-PCA treatment and the statistical results; e&f are the flow cytometric analysis and quantitative analysis of the infiltration level of MDSCs in the tumor after NanoICD / CAT-PCA treatment;

[0046] Figure 21 This is the immunofluorescence image of the expression level of HIF-1α in the tumor after NanoICD / CAT-PCA treatment;

[0047] Figure 22 These are the test results of NanoICD / CAT-PCA against tumor metastasis and recurrence, where a is the lung image of mice after treatment with PBS, nCAT-PCA, NanoICD-PCA and NanoICD / CAT-PCA; b is the tumor-free survival curve of mice in each group. DETAILED DESCRIPTION

[0048] The present invention provides a nanoparticle material capable of inducing cell immune death, comprising a functional protein core and a polymer layer in situ grown on the surface of the functional protein core, wherein the polymer monomers of the polymer layer include acrylamide, an endoplasmic reticulum targeting ligand, and a positively charged functional group compound;

[0049] The structural formula of the endoplasmic reticulum targeting ligand is shown in formula (1a) or formula (1b); the structural formula of the positively charged functional group compound is shown in any one of formulas (2) to (7);

[0050]

[0051] In the present invention, the functional protein core is preferably one or more of serum albumin, urate oxidase, catalase, glucose oxidase, horseradish peroxidase, superoxide dismutase, lactate dehydrogenase, acetaldehyde dehydrogenase, ethanol oxidase, and acetaldehyde oxidase. In the present invention, the enzymes in the functional protein core can degrade small molecule substrates associated with immune tolerance in the tumor microenvironment, thereby enhancing the synergistic anti-tumor effect with NanoICD.

[0052] In the present invention, the particle size of the nanoparticle material capable of inducing cell immune death is preferably 10 to 50 nm, more preferably 20 to 40 nm. In the present invention, the thickness of the polymer layer in the nanoparticle material capable of inducing cell immune death is preferably 5 to 20 nm, more preferably 10 nm.

[0053] In the present invention, the method for preparing the endoplasmic reticulum targeting ligand having the structure represented by formula (1) preferably comprises the following steps:

[0054] Under the action of an acid-binding agent, a compound having a structure shown in formula a undergoes a condensation reaction with (meth)acryloyl chloride to obtain an endoplasmic reticulum targeting ligand;

[0055]

[0056] In the present invention, the acid binding agent is preferably triethylamine; in the present invention, the molar ratio of the compound having the structure represented by formula a to (meth)acryloyl chloride is preferably 1:1.1 to 1.5, more preferably 1:1.2 to 1.4; the molar ratio of the acid binding agent to (meth)acryloyl chloride is preferably 1:1.

[0057] In the present invention, the organic solvent used in the condensation reaction is preferably ultra-dry dichloromethane; in the present invention, the condensation reaction is preferably carried out in an ice-water bath, and the reaction time is preferably 2 h.

[0058] After the condensation reaction, the present invention preferably performs column chromatography purification on the obtained condensation reaction product.

[0059] In the present invention, the synthesis route of the compound having the structure shown in formula a is shown in formula b:

[0060]

[0061] In the present invention, the positively charged functional group compound is commercially available.

[0062] The present invention provides a method for preparing the above-mentioned nanoparticle material capable of inducing cell immune death, comprising the following steps:

[0063] The buffer solution of the functional protein is mixed with N-acryloyloxysuccinimide to carry out a grafting reaction to obtain a double-bond functionalized protein;

[0064] The buffer solution of the double-bond functionalized protein is mixed with acrylamide, an endoplasmic reticulum targeting ligand, a positively charged functional group compound, a cross-linking agent and an initiator to carry out a polymerization reaction to obtain a nanoparticle material capable of inducing cell immune death.

[0065] The present invention mixes a buffer solution of a functional protein with N-acryloyloxysuccinimide, performs a grafting reaction, and obtains a double-bond functionalized protein.

[0066] In the present invention, the buffer solution of the functional protein is preferably a sodium bicarbonate buffer of the functional protein. In the present invention, the pH value of the sodium bicarbonate buffer is preferably 8.5, and the concentration is preferably 50 mM. In the present invention, the concentration of the buffer solution of the functional protein is preferably 5 to 50 mg / mL, more preferably 10 to 30 mg / mL.

[0067] In the present invention, the molar ratio of the N-acryloyloxysuccinimide to the functional protein is preferably 20 to 100:1, more preferably 40 to 80:1.

[0068] The present invention has no particular requirements for the mixing method; any mixing method known to those skilled in the art can be used, such as stirring. In the present invention, the grafting reaction temperature is preferably 4 to 37°C, more preferably 10 to 30°C, and the time is preferably 1 to 24 hours, more preferably 5 to 15 hours. The present invention introduces double bonds onto the surface of the functional protein through the grafting reaction.

[0069] After the grafting reaction, the present invention preferably separates the unreacted N-acryloyloxysuccinimide in the grafting reaction product. The method of removing the unreacted product is preferably one or more of dialysis, ultrafiltration, and desalination.

[0070] After obtaining the double-bond functionalized protein, the present invention mixes the buffer solution of the double-bond functionalized protein with acrylamide, an endoplasmic reticulum targeting ligand, a positively charged functional group compound, a cross-linking agent and an initiator to carry out a polymerization reaction to obtain a nanoparticle material that can induce cell immune death. In the present invention, the cross-linking agent is preferably N,N'-methylenebisacrylamide; the initiator is preferably ammonium persulfate and tetramethylethylenediamine, and the mass ratio of ammonium persulfate to tetramethylethylenediamine is preferably 1:2.

[0071] In the present invention, the concentration of the double-bond functionalized protein buffer solution is preferably 1 mg / mL. In the present invention, the endoplasmic reticulum targeting ligand, positively charged functional group compound and cross-linking agent are preferably added in the form of DMSO solution, and the acrylamide and initiator are preferably added in the form of aqueous solution.

[0072] In the present invention, the molar ratio of the functional protein to acrylamide, endoplasmic reticulum targeting ligand, and positively charged functional group compound is preferably 1:(2000-5000):(150-250):(200-1000), more preferably 1:3000:200:500; the molar ratio of the functional protein to the cross-linking agent is preferably 1:300, and the molar ratio of the functional protein to ammonium persulfate is preferably 1:300.

[0073] In the present invention, the polymerization reaction temperature is preferably 4 to 37° C., more preferably 10 to 30° C.; and the polymerization reaction time is 1 to 24 hours, more preferably 5 to 15 hours.

[0074] After the polymerization reaction, the present invention preferably separates and removes unreacted protein, cross-linking agent and initiator in the obtained polymerization product, and the separation method is preferably one or more of dialysis, ultrafiltration and desalination.

[0075] The present invention provides the use of the aforementioned nanoparticle material capable of inducing immune cell death in the preparation of an anti-tumor drug. The nanoparticle material capable of inducing immune cell death provided by the present invention can be used as an ICD inducer in tumor immunotherapy. In the present invention, the anti-tumor drug is preferably one or more of an anti-breast cancer drug, an anti-colon cancer drug, an anti-melanoma drug, an anti-glioma drug, an anti-uterine cancer drug, an anti-liver cancer drug, and an anti-pancreatic cancer drug.

[0076] In the present invention, the preparation process and functional diagram of the nanoparticle material capable of inducing cell immune death are as follows: Figure 1 shown.

[0077] The nanoparticle material capable of inducing cell immune death, its preparation method and application provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1 Preparation of NanoICD

[0079] 1) Synthesis of ETL

[0080] First, the precursor compound represented by Formula (a) was dissolved in ultra-dry dichloromethane. Acryloyl chloride or methacryloyl chloride was then added dropwise to the dichloromethane solution at a 1.1:1 molar ratio. Triethylamine was used as an acidifying agent at a 1:1 molar ratio to acryloyl chloride or methacryloyl chloride. The reaction was carried out at 4°C for 2 hours, followed by purification using standard column chromatography. The above-mentioned ETL compound 1 was obtained in a 95% yield.

[0081]

[0082] 1 H NMR(400MHz,DMSO-d6,δ):8.16(t,J=5.9Hz,1H,-CO-NH-CH2),7.69(t,1H,-CH2-NH-SO2-),7.67(d,J=7.9Hz,2 H,Ar-H),7.41(d,J=7.9Hz,2H,Ar-H),6.17(dd,J=17.1,10.0Hz,1H,CH2=CH-),6.06(dd,J=17.1,2.4Hz,1H,CH 1 H 2 =CH-),5.58(dd,J=9.9,2.4Hz,1H,CH 1 H 2=CH-), 3.17(q,J=6.5Hz,2H,-SO2-NH-CH2-), 2.78(q,J=6.6Hz,2H,-CO-NH-CH2-), 2.39(s,3H,-CH3)ppm.

[0083] 13 C NMR (400MHz, DMSO-d6, δ): 166.56, 143.72, 136.57, 130.58, 129.91, 127.09, 126.96, 43.05, 39.47, 21.58ppm.

[0084] MS:[M+Cl]:m / z:calcd.for(C 12 H 16 N2O3SCl):303.06, found 303.0573.

[0085] 2) Preparation of NanoICD

[0086] First, the protein was dissolved in 50 mM sodium bicarbonate buffer at pH 8.5 to prepare a 5 mg / mL protein solution. N-acryloyloxysuccinimide (NAS) was added and stirred at 4°C for 1 hour to ensure that double bonds available for polymerization were grafted onto the protein surface. The molar ratio of NAS to protein was 20:1. Unmodified NAS was removed by standard separation methods such as dialysis, ultrafiltration, and desalting.

[0087] Prepare DMSO solutions of ETL, PG and crosslinker N,N'-methylenebisacrylamide (BIS), prepare aqueous solutions of polymerization monomer acrylamide (Aam), initiators ammonium persulfate (APS) and tetramethylethylenediamine (TEMED);

[0088] To a 1 mg / mL protein solution, add Aam, ETL, PG, BIS, APS, and TEMED. The molar ratio of each component to protein is: Aam:ETL:PG:BIS:APS = 1:3000:200:500:300:300. The mass ratio of APS to TEMED is 1:2. The reaction is continued at 4°C for 1 hour. Unreacted protein, crosslinker, and initiator are removed by standard separation methods.

[0089] Structural characterization

[0090] (1) Determination of NanoICD particle size and morphology

[0091] Experimental Procedure: First, ultrasonically clean the Supelco sample vial for 30 minutes (adding a small amount of laundry detergent, three times), then rinse with double-distilled water until no visible buildup is observed. Seal the vial with aluminum foil and bake in an oven. After 24 hours, remove the dried light scattering vial, discard the foil, and soak in freshly prepared chromic acid solution for 24 hours. Rinse with double-distilled water until no visible buildup is observed. Seal the vial with aluminum foil and bake in an oven. After 24 hours, remove the dried light scattering vial, discard the foil, rinse in an acetone bath for 15 minutes, seal the vial with aluminum foil, and bake in an oven. After 24 hours, remove the dried light scattering vial and invert it for later use. Prior to particle size analysis, a NanoICD solution (phosphate buffer PB, pH 7.4, 10 mM) was freely dripped into the pretreated, clean light scattering vial using a Millipore syringe filter (220 nm). The particle size and distribution of the nanoparticles were then characterized.

[0092] The TEM sample preparation method is as follows: First, a carbon-coated copper grid is treated with gray light discharge to impart a positive surface charge to the grid, facilitating nanomaterial adsorption. A NanoICD solution is then dripped onto the grid, left to stand for 40 seconds, and excess liquid removed. The grid is then placed in a drop of 2% uranyl acetate solution for 30 seconds, repeated three times. After drying at room temperature, the sample morphology is observed using a Talos F200C transmission electron microscope (TEM) at an accelerating voltage of 120 kV.

[0093] The particle size and TEM image of NanoICD are as follows Figure 2 As shown by Figure 2 It can be seen that the particle size of NanoICD is about 20 nm and is spherical and evenly distributed.

[0094] (2) Determination of ETL content on the NanoICD surface

[0095] Experimental Procedure: First, ETL was dissolved in DMSO to prepare a 100 mM stock solution. Standard solutions ranging from 68.5 to 2192.2 μM were then serially diluted in PBS. UV absorbance of ETL at 235 nm was measured to establish a standard curve. UV absorbance of nBSA and NanoICD at 7.5 μM protein concentration was measured at 235 nm, and the ETL content on the surface of each NanoICD was determined by the difference calculation method.

[0096] The test results are as follows Figure 3 As shown in the figure, when the ETL feed ratio is 50, 100, 150 and 200, the ETL content on the NanoICD surface is 10, 20, 30 and 40 respectively. The different ETL contents on the NanoICD surface provide a solid basis for studying the nano-multivalent effect between NanoICD and the endoplasmic reticulum.

[0097] Performance Test 1

[0098] (1) Determination of NanoICD-induced calreticulin externalization in B16F10 cells

[0099] The operation steps are as follows: First, B16F10 cells were cultured at a rate of 1×10 5 Cells were seeded at a density of 10 cells / well in 12-well plates. After overnight incubation, the medium was replaced with fresh medium containing PTX (15 μM), ETL (100 μM), nBSA (1.5 μM), and NanoICD / BSA-n (n=10, 20, 30, 40; 1.5 μM). After a 12-hour incubation, cells were rinsed with PBS and incubated with ATTO488-conjugated antibodies and propidium iodide (PI) according to the manufacturer's instructions for flow cytometry analysis.

[0100] The test results are as follows Figure 4 As shown, NanoICD / BSA-30 and NanoICD / BSA-40 could effectively induce CRT externalization, while nBSA, NanoICD / BSA-10, and NanoICD / BSA-20 could not, indicating that the ETL density on the NanoICD surface is crucial for the induction of CRT externalization.

[0101] (2) Determination of NanoICD intracellular distribution

[0102] The operation steps are as follows: First, B16F10 cells were cultured at 2×10 4 Cells were seeded at a density of 10 cells / well in a 35 mm confocal culture dish (Φ = 15 mm). After overnight incubation, the culture medium was replaced with fresh medium containing nBSA (1.5 μM) and NanoICD / BSA-n (n = 10, 20, 30, 40; 1.5 μM). After incubation for 2, 4, 6, and 8 hours, the endoplasmic reticulum was labeled with a commercially available probe. The cells were then fixed and stained with DAPI. The cells were then photographed using a confocal laser scanner.

[0103] The test results are as follows Figure 5 As shown in (a), nBSA and NanoICD / BSA-10 are primarily distributed in the cytoplasm. Cells treated with NanoICD / BSA-20 exhibited significant colocalization in the ER at 4 hours, but at 6 and 8 hours, NanoICD / BSA-20 was transported back into the cytoplasm, and the colocalization signal decreased significantly. This suggests that NanoICD / BSA-20 failed to effectively overcome the retrograde transduction effect of the ER. In contrast, NanoICD / BSA-30 and NanoICD / BSA-40 were effectively enriched in the ER, with their accumulation increasing over time.

[0104] The colocalization coefficient was randomly analyzed using the Manders coefficient M2, and the results were as follows: Figure 5 As shown in (b), the M2 of cells treated with nBSA and NanoICD / BSA-10 was below 0.6 at all four time points, indicating that the low surface density of ETL (<10 per nanoparticle) was insufficient for efficient ER targeting of NanoICD. Cells treated with NanoICD / BSA-20 exhibited a higher M2 (0.82) at 4 h, but gradually decreased to 0.49 after 8 h of incubation, indicating that a moderate density of ETL (approximately 20 per nanoparticle) enhanced the interaction of NanoICD with the ER, but the interaction was insufficient to overcome reverse translocation and achieve long-term retention and accumulation in the ER. Notably, the M2 of cells treated with NanoICD / BSA-30 (0.86 at 6 h and 0.90 at 8 h) and NanoICD / BSA-40 (0.86 at 6 h and 0.95 at 8 h) were significantly higher than those of the other treatments and increased along with the incubation time, indicating that NanoICD / BSA requires a high density of ETL (>30 per nanoparticle) to effectively interact with the ER, overcome the reverse translocation that is retained in the ER, and ultimately induce ICD in the cells.

[0105] (3) Determination of the binding ability of NanoICD to the endoplasmic reticulum

[0106] Experimental Procedure: First, nBSA, NanoICD / BSA-20, and NanoICD / BSA-40 were attached to a gold chip on a QCM-D device. The interaction between NanoICD / CAT-n and the ER was characterized by mass changes and frequency shifts induced by the passage of isotonic extraction buffer containing ER. The specific procedure was as follows: ER extraction from mouse liver tissue: Fresh liver tissue was obtained from C57BL / 6 mice that had been starved overnight. The tissue was washed three times with cold PBS to remove blood, then cut into 1.5-2 cm pieces, repeatedly washed, blotted dry with a paper towel, and weighed to 1.28 g. The tissue was then cut into 0.3-0.5 cm pieces and placed in a glass homogenizer. 4.48 mL of 1× isotonic extraction buffer (3.5 mL per gram of tissue) was added and homogenized using a glass pestle. The homogenizer and pestle were then rinsed with 0.64 mL of 1× isotonic extraction buffer and added to the homogenized slurry. The homogenate was centrifuged at 1000×g for 10 minutes. The thin, floating lipid layer was gently aspirated and the supernatant was transferred to another centrifuge tube using a pipette. The supernatant, the post-mitochondrial fraction (PMF), was then centrifuged at 12,000×g for 15 minutes at 4°C. 4.6 mL of PMF was added to a beaker with a magnetic rod. 34.5 mL of 8 mM calcium chloride solution was slowly added dropwise. After complete addition, the mixture was stirred in an ice-water bath for 15 minutes. The mixture was collected and centrifuged at 8,000×g for 10 minutes. The pellet was homogenized and resuspended in 1× isotonic extraction buffer to obtain the rough endoplasmic reticulum. The QCM-D gold chip was pretreated by immersing the gold chip in a solution of deionized water: ammonia: 30% hydrogen peroxide (5:1:1 v / v / v) for 10 minutes in an ice-water bath for activation. The chip was then rinsed with ultrapure water and dried under nitrogen. A 100 μg / mL solution of 2-iminothiolane hydrochloride (Traut's Reagent) and tris(2-carboxyethyl)phosphate hydrochloride (TCEP) nanocapsules was added to the activated gold chip and incubated at room temperature for 24 hours. After the reaction, the gold chip was carefully removed, rinsed with ultrapure water, and dried under nitrogen to obtain a gold chip modified with NanoICD and nBSA. The modified gold chip was placed in the detection chamber and phosphate-buffered saline (PBS) was introduced at a flow rate of 10 μL / min until a stable baseline was achieved. An isotonic extraction buffer containing ER was then introduced at a flow rate of 2 μL / min for 30 minutes. Finally, ultrapure water and air were introduced at a flow rate of 10 μL / min to rinse the gold chip. During the test, the sample inlet tube was kept free of bubbles and kept at a constant temperature of 37°C.

[0107] The experimental results are as follows Figure 6As shown, NanoICD / BSA-40 (ΔF = 149.85 Hz) exhibited a significantly higher maximum frequency shift than NanoICD / BSA-20 (ΔF = 110.14 Hz) and nBSA (ΔF = 91.68 Hz), indicating that the high-density ETL imparted a strong binding affinity between NanoICD / BSA-40 and the ER. More importantly, when washed with PBS, the binding affinity between NanoICD / BSA-40 and the ER was significantly improved compared with the NanoICD / BSA-20 (ΔF = 68.2 Hz, ΔΔF = 41.94 Hz) and nBSA groups (ΔF = 7 Hz, ΔΔF = 84.67 Hz), indicating that the strong binding of NanoICD / BSA-40 provides the potential to overcome retrotranslocation and remain in the ER.

[0108] (4) Determination of NanoICD-induced HMGB-1 efflux

[0109] The operation steps are as follows: First, B16F10 cells were cultured at 2×10 4 Cells were seeded at a density of 100 cells / well in a 35 mm confocal culture dish (Φ = 15 mm). After overnight incubation, the medium was replaced with fresh medium containing PTX, ETL, nBSA (1.5 μM), and NanoICD / BSA. After an 8-hour incubation, the cells were washed with cold PBS, fixed with 4% paraformaldehyde for 15 minutes, and rinsed with cold PBS. The membrane was then permeabilized with 0.1% Triton X-100 for 10 minutes, followed by another PBS wash. The cells were then blocked with 5% BSA blocking buffer for 1 hour at room temperature, and then an HMGB-1 primary antibody was added, followed by overnight incubation at 4°C in the dark. Excess primary antibody was then washed with cold PBS, and an Alexa 647-conjugated goat anti-rabbit secondary antibody was added, followed by incubation at room temperature in the dark for 2 hours. After PBS washing, cell nuclei were stained with DAPI for CLSM observation. The HMGB-1 concentration in the cell supernatant was determined using the corresponding ELISA kit.

[0110] The test results are as follows Figure 7 As shown in the figure, after treatment with PTX and NanoICD / BSA, HMGB-1 efficiently effluxed from the nucleus of cells, indicating that NanoICD / BSA can effectively induce cells to undergo ICD. In addition, the concentration of HMGB-1 in the supernatant was also effectively upregulated.

[0111] (5) NanoICD / BSA-induced EIF2a phosphorylation experiment

[0112] Flow cytometry and Western blot analysis were used to investigate the NanoICD / BSA-induced EIF2a phosphorylation experiment. Flow cytometry was performed as follows: B16F10 cells were plated at 1×10 5The cells were seeded at a density of 10 cells / well in a 12-well plate for one day, and then PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM) and NanoICD / BSA (1.5 μM) were added to the cells. After incubation for 12 hours, the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes. Nonspecific binding sites were pre-incubated with PBS containing 5% FBS for 30 minutes, followed by incubation with the primary antibody for 1 hour, and then washed three times with PBS and incubated with Alexa488-conjugated monoclonal secondary antibody for 30 minutes, and then used for flow cytometry analysis. The results are shown in Figure 2. Figure 8 As shown in (a), after treatment with NanoICD / BSA and PTX, the expression level of pEIF2a in cells was significantly upregulated, indicating that NanoICD / BSA effectively generated endoplasmic reticulum stress.

[0113] Western blotting was performed as follows: B16F10 cells were plated at 2 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates overnight and then treated with PBS, PTX (15 μM), ETL (100 μM), nBSA (1.5 μM) and NanoICD / BSA (1.5 μM) for 24 hours. After incubation, the cells were rinsed with PBS and dissolved in 1% Nonidet P-40 lysis buffer. The homogenate was clarified by centrifugation at 20,000 g for 15 minutes at 4°C, and the protein concentration was determined using the BCA assay. Total protein lysates were separated by SDS-PAGE on 10% SDS acrylamide gels and then transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies to EIF2α, pEIF2α and β-actin (1:1000 dilution) overnight and then incubated with HRP-conjugated secondary antibodies (1:2000 dilution) for 1 hour. The results are shown in Figure 2. Figure 8 As shown in middle b, after treatment with NanoICD / BSA and PTX, the expression level of pEIF2a in cells was significantly upregulated, indicating that NanoICD / BSA effectively generated endoplasmic reticulum stress.

[0114] (6) RNAseq analysis

[0115] B16F10 cells were grown at 5×10 6 Cells were seeded in culture dishes at a density of 100 cells / well for one day, and then PBS, nBSA (1.5 μM), and NanoICD / BSA (1.5 μM) were added to the cells. After incubation for 24 hours, the cells were rinsed with cold PBS and then subjected to RNAseq analysis.

[0116] The results are as follows Figure 9As shown, the endoplasmic reticulum unfolded protein response (ER UPR), immune system process, response to ER stress, and response to unfolded protein pathway were the most upregulated GO terms in NanoICD-treated cells, and protein processing in the endoplasmic reticulum and antigen processing and presentation pathway were the most upregulated KEGG terms. These upregulated gene terms suggest that NanoICD / BSA, as a biomimetic unfolded / misfolded protein, can induce ER stress and ER UPR, promote antigen processing and presentation, and ultimately activate immune system processes.

[0117] (7) NanoICD / BSA activates the anti-tumor immune system in vitro

[0118] Isolation and Culture of Bone Marrow-Derived Dendritic Cells: 6-8 week-old C57BL / 6 mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 15-25 minutes. The cells were then transferred to a clean bench. The hind limbs of the mice were amputated using sterile scissors, and surrounding muscle tissue was carefully removed. The tibia and fibula were then cut with scissors, and the epiphyses at both ends were removed. An appropriate amount of PBS was drawn up with a syringe and the needle was inserted into the bone marrow cavity to flush the bone marrow until it turned white. The collected bone marrow suspension was filtered through a 200-mesh nylon mesh into a centrifuge tube. After centrifugation, the supernatant was discarded and 8 mL of red blood cell lysis buffer was added. The cells were lysed for 10 minutes and then centrifuged. The cells were resuspended in 1640 medium supplemented with 10% FBS and plated evenly in 6-well plates. On days 2 and 4, the medium was changed by half, with 1640 medium supplemented with 10 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). On the 5th day, BMDCs were purified by gradient density centrifugation and used in subsequent experiments.

[0119] NanoICD / BSA induced dendritic cell maturation: Flow cytometry was used to detect the maturation of BMDCs induced by NanoICD / BSA. Purified BMDCs were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate, and then B16F10 cells treated with PBS, PTX, ETL, nBSA and NanoICD / BSA were added to BMDC and incubated for 24 hours. After collecting the cells, centrifugation was performed and the supernatant was discarded. After resuspending with PBS, anti-CD11C-APC, anti-CD80-FITC, and anti-CD86-PE antibodies were added and stained at 4°C in the dark for 2 hours. After staining, the cells were centrifuged and washed with cold PBS to remove unbound antibodies. After fixation with 4% paraformaldehyde, the BMDC maturation ratio was detected by an instrument. The experimental results are shown in the figure. Figure 10 As shown in (a), PTX and NanoICD / BSA treatment can effectively induce dendritic cell maturation.

[0120] NanoICD / BSA induces dendritic cells to phagocytose tumor cells: Flow cytometry was used to detect the phagocytic effect of NanoICD / BSA on BMDCs on tumor cells. The B16F10 cell suspension was stained with DiD dye in a cell culture incubator for 15 minutes, washed with PBS, and evenly seeded in a 6-well plate. After culturing for 24 hours, PBS, PTX, ETL, nBSA and NanoICD / BSA were added to the cells and cultured for another 12 hours. The B16F10 cells were then incubated with BMDCs that had been pre-stained with DiO dye for 12 hours. After the culture was completed, the cells were collected, centrifuged, washed with cold PBS, and fixed with 4% paraformaldehyde. The phagocytosis of B16F10 by BMDCs was detected by flow cytometry (FITC+APC+). The experimental results are shown in the figure. Figure 10 As shown in middle b, PTX and NanoICD / BSA treatment can effectively induce dendritic phagocytosis of tumor cells.

[0121] NanoICD / BSA promotes dendritic cell antigen presentation: NanoICD / BSA induced BMDC antigen presentation to tumor cells was detected by flow cytometry. The purified BMDC was seeded in a 6-well plate at a cell density of 2×105 / well, and then B16F10-OVA cells treated with PBS, PTX, ETL, nBSA and NanoICD / BSA were added to the BMDC and incubated for 24 hours. After collecting the cells, centrifuge and discard the supernatant, resuspend with PBS, add anti-CD11C-APC and anti-MHC-I-FITC antibodies and stain at 4°C in the dark for 2 hours. After staining, centrifuge and wash with cold PBS to remove unbound antibodies, fix with 4% paraformaldehyde and detect BMDC antigen presentation on the machine. The experimental results are as follows Figure 10 As shown in (c), PTX and NanoICD / BSA treatment can effectively promote dendritic cell antigen presentation.

[0122] (8) NanoICD / BSA inhibits tumor growth

[0123] 1×10 6 B16F10 cells were orthotopically injected into the mammary glands of 6-8 week-old female C57BL / 6 mice until the tumor size reached 100 mm. 3 Tumor size was measured with a vernier caliper, and tumor volume was calculated using the following formula: V = W 2×L / 2, where W and L are the shortest and longest diameters of the tumor, respectively. The mice were randomly divided into five groups: PBS, PTX, ETL, nBSA and NanoICD / BSA, with 6 mice in each group. The tumors were injected orally with drugs (200μL), PTX (15μM), ETL (100μM), nBSA (1.5μM) and NanoICD / BSA (1.5μM). The drugs were given once every three days for a total of 5 times. The tumor volume of the mice was measured for 22 days starting from the first day of administration. Figure 11 As shown, compared with the control group, the NanoICD / BSA group showed a slower tumor growth rate and a short-term inhibitory effect after drug cessation, demonstrating the excellent tumor inhibition effect of NanoICD / BSA. Monitoring the weight of mice showed no significant toxicity in all five groups.

[0124] (9) NanoICD / BSA activation in vivo promotes CRT externalization

[0125] The experimental operation of flow cytometer analysis was as follows: the tumors of mice after the third administration of the drug were collected, the tumor tissues were cut into small pieces, ground into single cell suspensions using a homogenizer, and then 70×10 -6 m cell strainer. Then dilute the filtered cell suspension to 1×10 7 cells / mL, add fluorescently labeled antibodies and incubate at 4℃ in the dark for 2 hours. After staining, centrifuge to remove excess antibodies and then wash with cold PBS for flow cytometry analysis. Figure 12 As shown, NanoICD / BSA can effectively induce CRT eversion in vivo.

[0126] (10) NanoICD / BSA activates ICD-related anti-tumor immunity in vivo

[0127] The tumors of mice after the third treatment were collected, the tumor tissues were cut into small pieces, ground into single cell suspension using a homogenizer, and then 70×10 -6 m cell strainer. Then dilute the filtered cell suspension to 1×10 7 Cells / mL were added with fluorescently labeled antibodies (APC-CD11C, FITC-CD80, PE-CD86) and incubated at 4°C in the dark for 2 hours. After staining, the cells were centrifuged to remove excess antibodies and washed with cold PBS. After fixation with 4% paraformaldehyde, the cells were used for flow cytometry analysis of NanoICD / BSA-promoted DC maturation. The results are shown in Figure 2. Figure 13 As shown in (a), NanoICD / BSA can effectively promote DC maturation.

[0128] The tumors of mice after the third treatment were collected, the tumor tissues were cut into small pieces, ground into single cell suspension using a homogenizer, and then 70×10-6 m cell strainer. Then dilute the filtered cell suspension to 1×10 7 Cells / mL were added with fluorescently labeled antibodies (APC-CD3, PE-CD8, APC-Cy7-CD62L, FITC-CD44) and incubated at 4°C in the dark for 2 hours. After staining, the cells were centrifuged to remove excess antibodies and washed with cold PBS. The cells were then fixed with 4% paraformaldehyde and analyzed by flow cytometry for the generation of memory T cells promoted by NanoICD / BSA. The results are shown in Figure 2. Figure 13 As shown in (a), NanoICD / BSA can promote the generation of memory T cells.

[0129] The above results indicate that NanoICD / BSA can effectively activate ICD in vivo and trigger ICD-related anti-tumor immune responses.

[0130] (11) NanoICD / BSA promotes T cell tumor infiltration, T cell proliferation, and T cell tumor killing

[0131] Freshly harvested tumor tissue was cut into small pieces and homogenized using a GentleMACs Dissociator, followed by 70 × 10 -6 Filter the tumor homogenate through a cell strainer to prepare a single cell suspension. Collect the cells and dilute to 1×10 7 cells / mL. Add fluorescent antibody-conjugated primary antibody to the cell suspension and incubate at room temperature for 30 minutes. After staining, the cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry. Figure 14 As shown in a, NanoICD / BSA can effectively increase T cell infiltration in tumors; Figure 14 As shown in b, NanoICD / BSA can effectively maintain the proliferation ability of T cells; Figure 14 As shown in Figure c, NanoICD / BSA can effectively enhance the anti-tumor activity of T cells. The above results indicate that NanoICD / BSA can effectively regulate the tumor microenvironment in mice.

[0132] (12) NanoICD / BSA inhibits tumor recurrence

[0133] First, B16F10 cells were pretreated with PTX, ETL, nBSA and NanoICD / BSA for 24 h, and then 1×10 6 Cells were inoculated into the left groin of 6-8 week old C57BL / 6 mice. 7 days later, 1×10 5 Untreated tumor cells of the same type were then used to continuously monitor the growth of tumors on the right side of the mice over 60 days. Figure 15 As shown, NanoICD / BSA can effectively inhibit tumor recurrence.

[0134] (13) NanoICD / BSA inhibits tumor metastasis

[0135] First, B16F10 cells were pretreated with PTX, ETL, nBSA and NanoICD / BSA for 24 h, and then 1×10 6 Cells were inoculated into the left groin of 6-8 week old C57BL / 6 mice. 7 days later, 1×10 5 The mice were killed 10 days later and the lung metastasis of the tumor was observed. Figure 16 As shown in the results, NanoICD / BSA can effectively inhibit tumor metastasis to the lungs.

[0136] Example 2 Synthesis of NanoICD / CAT-PCA

[0137] ① Modification of catalase (CAT): Prior to polymerization, catalase double bonds were modified with N-acryloyloxysuccinimide (NAS) to provide sites for in situ polymerization. The specific steps were as follows. First, 100 mg of CAT was weighed into a round-bottom flask and dissolved in 100 mM sodium bicarbonate buffer with stirring to adjust the concentration to 10 mg / mL. NAS (dissolved in ultra-dry DMSO to make a 30 mg / mL stock solution) was then added dropwise to the solution, with a CAT:NAS molar ratio of 1:20. The reaction was stirred at 4°C for 2 h, followed by centrifugal ultrafiltration to remove unreacted NAS (4°C, 5000 rpm). The acryloylated CAT was standardized to a certain concentration and stored in a refrigerator at 4°C for subsequent experiments.

[0138] Preparation of NanoICD / CAT: First, dissolve acrylamide (AAm) in ultrapure water to prepare a 20% (w / v) stock solution, dissolve N-(3-aminopropyl)acrylamide (Apm) in ultrapure water to prepare a 10% stock solution, and dissolve ETL and N,N-methylenebisacrylamide (Bis) in ultra-dry DMSO to prepare a 10% stock solution. Place 2 mg of acryloylated CAT in a 2 mL centrifuge tube. Add monomers AAm, Apm, and ETL, followed by the crosslinker Bis, and mix thoroughly. (The optimal molar ratio of CAT / AAm / Apm / ETL / Bis is 1 / 3500 / 550 / 200 / 420; other polymerization ratios include 1 / 3500 / 550 / 0 / 420; 1 / 3500 / 550 / 100 / 420; and 1 / 3500 / 550 / 150 / 420.) Then, initiators tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) (APS / CAT = 350:1, n / n; TEMED / APS = 2:1, w / w) were added, and the reaction solution concentration was adjusted to 2 mg / mL using sodium bicarbonate buffer. The reaction was allowed to react at 4°C for 2 h. The reaction solution was centrifuged and concentrated using Millipore ultrafiltration tubes (MWCO = 100 kDa) to remove unreacted impurities, yielding protein nanocapsules (NanoICD / CAT / NanoICD / CAT-n). After standardization, the solution was stored at 4°C until use.

[0139] ③ Preparation of NanoICD / CAT-PCA: NanoICD / CAT-PCA is formed by the electrostatic interaction between NanoICD / CAT and the block copolymer mPEG113-b-PLys100 / CA. mPEG113-b-PLys100 / CA was prepared according to a previously published article (Adv. Mater. 2022, 34, 2201945). First, mPEG113-b-PLys100 / CA was dissolved in sodium bicarbonate buffer. The NanoICD / CAT solution was then mixed with the mPEG113-b-PLys100 / CA solution at a ratio of 1:10 (m / m). Immunomodulatory nanoparticles (NanoICD / CAT-PCA) were obtained by centrifugal ultrafiltration. After calibration, the mixture was stored at 4°C for subsequent experimental use. By encapsulating NanoICD, PCA effectively prevents nonspecific clearance of NanoICD by the immune system in the bloodstream, improves NanoICD accumulation in tumors, and reduces toxic side effects.

[0140] Performance Test 2

[0141] (1) NanoICD / CAT-PCA enrichment ability in tumors after intravenous injection

[0142] First, 4T1 cells (5×10 6 / mouse), and wait until the tumor volume grows to 200mm 3 Mice were randomly divided into two groups. 100 μL of Cy5-NanoICD / CAT and Cy5-NanoICD / CAT-PCA were injected into the tail vein respectively. 24 and 48 hours after the tail vein injection, the mice were killed by dislocation, and the tumors and major organs such as the heart, liver, spleen, lung, and kidney were collected. The distribution of Cy5 fluorescent-labeled nanoparticles in the body was then observed using a small animal in vivo imaging device. Figure 17 As shown, NanoICD / CAT-PCA can be effectively enriched in tumor tissue after intravenous injection.

[0143] (2) In vivo study of NanoICD / CAT-PCA against tumor growth

[0144] First, 4T1 cells (5×10 6 / mouse) to construct a tumor-bearing mouse model. When the tumor grew to 50 mm 3 At the same time, tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group. At the same time, nanoparticles with only the function of catalyzing hydrogen peroxide (nCAT-PCA) and nanoparticles with only the function of targeting the endoplasmic reticulum (NanoICD-PCA) were synthesized according to the previous preparation method. 100 μL of nCAT-PCA, NanoICD-PCA and NanoICD / CAT-PCA (concentration of 1 mg / mL) were injected into the tail vein respectively. The PBS injection group served as the negative control group. The drug was given once every 3 days for 19 days. The diameter of the mouse tumor was measured with a vernier caliper and referred to the formula: V = d 2 ×D / 2 to calculate its volume, where d represents the shortest diameter of the tumor and D represents the longest diameter of the tumor, and the measurement unit is mm. At the same time, in order to evaluate the potential toxicity of nanoparticles, the weight of mice was also monitored during the tumor inhibition experiment. Figure 18 As shown in Figure a, nCAT-PCA has no significant advantage over PBS in tumor suppression in mice. In contrast, NanoICD-PCA and NanoICD / CAT-PCA can effectively inhibit tumor growth in mice, with mice treated with NanoICD / CAT-PCA showing a more significant tumor suppression effect. In addition, NanoICD / CAT-PCA also significantly improved the survival rate of 4T1 tumor-bearing mice. During the 35-day monitoring period, 50% of the tumor-bearing mice survived ( Figure 18 b). During the treatment, there was no significant change in the weight of the mice, indicating that the nanoparticles did not cause any significant toxic side effects on the mice ( Figure 18 c) in the above example.

[0145] (3) NanoICD / CAT-PCA promotes T cell infiltration

[0146] To evaluate the ability of NanoICD / CAT-PCA to activate the immune system of mice, flow cytometry and immunofluorescence staining were used to characterize the infiltration of effector T cells in the tumor. The experimental procedure for flow cytometry analysis was as follows: the tumors of mice after the third treatment were collected, the tumor tissues were cut into small pieces, ground into single cell suspensions using a homogenizer, and then 70×10 -6 m cell strainer. Then dilute the filtered cell suspension to 1×10 7 Cells were stained with fluorescently labeled antibodies at 4°C for 2 hours in the dark. After staining, sections were centrifuged to remove excess antibody and washed with cold PBS. Sections were fixed with 4% paraformaldehyde and analyzed by flow cytometry. Immunofluorescence staining was performed as follows: frozen sections were removed from the -80°C freezer, and an appropriate amount of PBS was added to cover the tissue. The sections were allowed to wet for 15 minutes, after which the PBS was removed and blocked with 5% BSA solution for 1 hour. Rabbit anti-mouse CD4 primary antibodies and rat anti-mouse CD8 primary antibodies were then added dropwise, and the sections were incubated overnight at 4°C in the dark. Following incubation, sections were washed three times with cold PBS, followed by the addition of corresponding Alexa 594-conjugated donkey anti-rabbit secondary antibodies and Alexa 488-conjugated goat anti-rat secondary antibodies, and incubated at 37°C in the dark for 2 hours. After staining, cell nuclei were stained with DAPI. After washing with PBS, sections were mounted with anti-fluorescence quenching mounting medium, and tumor-infiltrating T cells were observed using CLSM. Flow cytometry was also used to characterize the proliferation and cytotoxicity of infiltrating T cells. The cell suspension was prepared using the same method as described above. Cells were first fixed with 4% paraformaldehyde, then permeabilized with 0.1% Triton X-100 for 10 minutes and washed twice with cold PBS. Antibodies were then added to the cell suspension. After staining, unbound antibodies were removed by centrifugation, and the cells were thoroughly washed with cold PBS before analysis using a flow cytometer.

[0147] like Figure 19 As shown in Figures a and b, mice treated with nCAT-PCA (15.6%) and NanoICD-PCA (15.9%) showed lower levels of CD8+ T cell infiltration compared to the PBS group (6.28%). However, the number of infiltrating CD8+ T cells in the tumor tissue of mice treated with NanoICD / CAT-PCA significantly increased, reaching a proportion of 45.7%. This result indicates that NanoICD / CAT-PCA can effectively promote the infiltration of effector T cells. Figure 19The immunofluorescence staining results in c also showed that a small amount of CD8+T fluorescence signals were observed in nCAT-PCA and NanoICD-PCA, and a small amount of CD4+T cells were observed in the NanoICD-PCA group. + T and CD4 + The T cell fluorescence signal increased significantly, indicating that the infiltration level of effector T cells was significantly improved.

[0148] (4) Research on NanoICD / CAT-PCA remodeling the immunosuppressive tumor microenvironment

[0149] The macrophage phenotype and immunosuppressive cell number in tumor tissue were analyzed by flow cytometry. The specific operation was as follows: the tumor of mice after the third treatment was collected, the tumor tissue was cut into small pieces, and it was ground into single cell suspension using a homogenizer. -6 m cell strainer. Then dilute the filtered cell suspension to 1×10 7 cells / mL, add fluorescently labeled antibodies (APC-Cy7-CD45, FITC-CD3, APC-CD4, PE-CD8) and incubate at 4°C in the dark for 2 hours. After staining, centrifuge to remove excess antibodies and then wash with cold PBS. Then fix with 4% paraformaldehyde and use for flow cytometric analysis. Figure 20 As shown in a to f, NanoICD / CAT-PCA treatment can effectively improve the expression of M2 macrophages in tumors and reduce the content of Tregs and MDSCs, indicating that NanoICD / CAT-PCA can effectively reshape the immunosuppressive tumor microenvironment.

[0150] The expression of hypoxia-inducible factor (HIF-1α) was characterized by immunofluorescence staining. The frozen sections were taken out from the -80℃ freezer, and an appropriate amount of PBS was added to the sections to cover the tissue. The sections were wet for 15 minutes, and then the PBS was shaken off. 0.1% TritonX-100 was added to break the membrane for 10 minutes, and then the sections were washed with PBS and blocked with 5% BSA solution for 1 hour. Then, HIF-1α primary antibody was added and incubated overnight at 4℃ in the dark. After the incubation, the corresponding Alexa594-coupled fluorescent secondary antibody was added after washing with cold PBS several times, and incubated at 37℃ in the dark. After 2 hours of incubation, the cell nuclei were stained with DAPI dye, and after washing with PBS, the sections were sealed with anti-fluorescence quenching sealing agent, and the expression of HIF-1α was observed using CLSM. As Figure 21As shown, after treatment with catalase-containing nCAT-PCA, the fluorescence signal of HIF-1α was significantly reduced compared to the PBS group, indicating that catalase catalyzes hydrogen peroxide to produce oxygen, which can effectively alleviate the hypoxic microenvironment. The expression level of HIF-1α in tumors of mice treated with NanoICD / CAT-PCA was also significantly reduced, indicating that NanoICD / CAT-PCA can effectively alleviate the hypoxic tumor microenvironment.

[0151] (5) Research on NanoICD / CAT-PCA against tumor metastasis and recurrence

[0152] Anti-metastasis study: 4T1 cells (1×10 6 / mouse), the tumor grew to 50mm 3 Tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group. Then, 100 μL of nCAT-PCA, NanoICD-PCA and NanoICD / CAT-PCA (final concentration of 1 mg / mL) were injected into the tail vein every two days for a total of 3 times. Then, 1×10 5 4T1 cells were used to construct a lung metastasis model. After 10 days, the mice were killed by cervical dislocation and their lung tissues were collected. After washing with PBS, the number of nodules was observed and photographed. Figure 22 As shown in a, the number of lung nodules in mice treated with NanoICD / CAT-PCA was significantly reduced, that is, there were no obvious tumor metastases, indicating that mice treated with NanoICD / CAT-PCA had a significant inhibitory effect on the lung metastasis of tumor cells.

[0153] Anti-relapse study: 4T1 cells (1×10 6 / mouse). When the tumor volume reached 50 mm3, the tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group. Then, 100 μL of nCAT-PCA, NanoICD-PCA and NanoICD / CAT-PCA (final concentration of 1 mg / mL) were injected into the tail vein once every two days for a total of 3 times. Then, 4T1 cells (1×10 5 / mouse) to construct a tumor recurrence model. During the experiment, the growth of the right side tumor of the mice was continuously observed and recorded. Figure 22As shown in (b), by day 28, all mice in the PBS control group had palpable tumors in the right mammary gland. Mice treated with nCAT-PCA began developing tumors in the right mammary gland on day 20, and all mice in this treatment group had tumors on the contralateral side by day 33, indicating a delay in tumor growth in the nCAT-PCA group. Furthermore, mice treated with NanoICD-PCA and NanoICD / CAT-PCA began developing right-sided tumors on days 24 and 26, respectively. By day 45, 16.7% and 33.3% of mice in these two treatment groups, respectively, had no tumors on the distal right side, demonstrating that NanoICD / CAT-PCA effectively inhibits tumor recurrence.

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nanoparticle material capable of inducing cell immune cell death, comprising a functional protein core and a polymer layer grown in situ on the surface of the functional protein core, wherein the polymer monomers of the polymer layer include acrylamide, an endoplasmic reticulum targeting ligand, and a positively charged functional group compound; The structural formula of the endoplasmic reticulum targeting ligand is shown in formula (1a) or formula (1b); the structural formula of the positively charged functional group compound is shown in any one of formulas (2) to (7); The functional protein core is serum albumin or catalase.

2. The nanoparticle material capable of inducing cell immune death according to claim 1, characterized in that: The particle size of the nanoparticle material capable of inducing cell immune death is 10 to 50 nm.

3. A method for preparing the nanoparticle material capable of inducing cell immune death according to claim 1 or 2, comprising the following steps: The buffer solution of the functional protein is mixed with N-acryloyloxysuccinimide to carry out a grafting reaction to obtain a double-bond functionalized protein; The buffer solution of the double-bond functionalized protein is mixed with acrylamide, an endoplasmic reticulum targeting ligand, a positively charged functional group compound, a cross-linking agent and an initiator to carry out a polymerization reaction to obtain a nanoparticle material capable of inducing cell immune death.

4. The preparation method according to claim 3, characterized in that The method for preparing the endoplasmic reticulum targeting ligand comprises the following steps: Under the action of an acid-binding agent, a compound having a structure shown in formula a undergoes a condensation reaction with (meth)acryloyl chloride to obtain an endoplasmic reticulum targeting ligand; 5. The preparation method according to claim 3, characterized in that The molar ratio of the N-acryloyloxysuccinimide to the functional protein is 20 to 100:1; The grafting reaction temperature is 4 to 37° C., and the time is 1 to 24 hours.

6. The preparation method according to claim 3, characterized in that The cross-linking agent is N,N'-methylenebisacrylamide; The initiators are ammonium persulfate and tetramethylethylenediamine.

7. The preparation method according to claim 3 or 6, characterized in that: The molar ratio of the functional protein to acrylamide, the endoplasmic reticulum targeting ligand, and the positively charged functional group compound is 1:(2000-5000):(150-250):(200-1000).

8. The preparation method according to claim 4, characterized in that The polymerization reaction temperature is 4 to 37° C., and the reaction time is 1 to 24 hours.

9. Use of the nanoparticle material capable of inducing cell immune death according to claim 1 or 2 or the nanoparticle material capable of inducing cell immune death prepared by the preparation method according to any one of claims 3 to 8 in the preparation of anti-tumor drugs.

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