Antibody-metal modified exosomes with anti-tumor immune enhancement effect and preparation method and application thereof

Abstract

The present invention belongs to the field of biotechnology and relates to an antibody-metal modified exosome with anti-tumor immune enhancement effect and a preparation method and application thereof. 2+ The introduction of a purely biological exosome delivery system has innovated the construction form of related delivery carriers; the present invention uses exosome delivery for drug delivery to improve drug tumor penetration; the introduction of Mn 2+ It works synergistically with PDL1 Nb and DOX, and the trinity improves the efficacy and reduces systemic toxicity. The exosomes transformed by the method of the present invention have good functional effects of killing tumors, inducing ICD and activating cGAS-STING to promote immune activation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an antibody-metal modified exosome with anti-tumor immune enhancement effect and a preparation method and application thereof. Background Art

[0002] Immunotherapy, especially the PDL1 / PD1 checkpoint blockade strategy, can relieve the tumor's immunosuppression of CTLs and enable them to kill tumors normally, and is considered to be one of the few strategies that may completely cure tumors. However, the overall efficacy of PD1 / PDL1 blockers is less than 30% at present, because the immunosuppressive tumor microenvironment (TME) formed by the tumor in the body lacks the infiltration of functional CTLs, resulting in a lack of soil for the drug to take effect. Therefore, considering how to further activate CTLs and promote their infiltration in the TME during the immune response is a top priority for optimizing PD1 / PDL1 checkpoint blockade therapy.

[0003] The anti-tumor immune response includes several essential steps: 1) the release of tumor-associated antigens after natural exposure / death of the tumor; 2) presenting cells such as dendritic cells (DCs), which take up and process antigens, complete their own maturation in the process, and finally present the antigens to downstream immature T cells; 3) immature T cells proliferate and differentiate into CD8 + T cells (i.e. CTLs) chemotaxis, infiltration and killing of tumor cells in the local tumor. In this process, unlike the action link of PD1 / PDL1 blockers, the anti-tumor drug doxorubicin (DOX) can induce immunogenic cell death (ICD) of tumor cells in the initial stage, further release and expose tumor-associated antigens, increase the activation level of downstream CTLs, and can be used in combination with blockers. However, direct use of DOX will produce dose-dependent cardiotoxicity, and it is necessary to consider how to limit its effect to the tumor site, or use auxiliary factors to reduce the immune activation burden of DOX and reduce the amount of DOX used. Mn 2+ Because it can promote DC maturation, amplifying the downstream effects of DOX becomes an option that meets the needs, but its neurotoxicity also points to the need for local effects. So far, considering the appropriate drug controlled release system to deliver multiple drugs (PD1 / PDL1 blockers, DOX, Mn 2+ ) is delivered to the local part of the tumor tissue, which has become a strategy to promote the combined effectiveness of various drugs and reduce toxic side effects. The current drug delivery carriers, mainly many artificial synthetic carriers, can induce adverse reactions such as allergies. Natural biological carriers are the source of ideas to solve this problem.

[0004] Exosomes are cell membrane-derived vesicles secreted by cells with a diameter of 30-150nm. They can carry regulatory factors and play a wide range of roles in the physiological and pathological processes of the human body. Therefore, they naturally have excellent biocompatibility and circulation stability. At present, the transformation of exosomes as a biomaterial and the use of a variety of imaging molecules, probes and therapeutic agents for in vivo applications have formed a unique "exosome diagnosis and treatment". Among them, the delivery of drugs to treat diseases with exosomes has produced a variety of research results, especially for the deep delivery of drugs into the layers of barriers of the microenvironment of solid tumors.

[0005] For loading multiple drug components simultaneously, although electroporation is a mature method to load DOX into exosomes, there are many gaps in the research on how to couple antibodies and metal ions into exosome delivery systems, especially the introduction of transition metal ions into pure biogenic systems. Some people have synthesized chelates of DOX and multiple metal ions, which provides the possibility of loading chelates by electroporation, but Mn 2+ The combination with commercially available doxorubicin hydrochloride failed due to the presence of hydrochloric acid in the system. Therefore, exploring a simple construction paradigm with the potential for large-scale preparation will help innovate the application of exosome drug delivery platforms. Based on this, this work prepared an antibody-metal modified exosome αPDL1@DOX-Mn with anti-tumor immune activation enhancement effect. 2+ , forming a simple and scalable construction model for introducing antibodies / metal ions into exosome delivery systems. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an antibody-metal modified exosome with anti-tumor immune enhancement effect and its preparation method and application, which utilizes exosome delivery for drug delivery, improves drug tumor penetration, and introduces Mn 2+ It works synergistically with PDL1 Nb and DOX, and the trinity improves efficacy and reduces systemic toxicity.

[0007] The technical solution provided by the present invention is as follows:

[0008] A method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect comprises the following steps:

[0009] The membrane anchored protein of the nanobody fragment containing the PDL1 targeting protein is transfected into HEK-293T cells, and the cell membrane transformed with the PDL1 Nb-GFP-TRP PK1 fusion protein is extracted after the protein is expressed; the amino acid sequence of the nanobody fragment containing the PDL1 targeting protein is shown in SEQ ID NO.1; the amino acid sequence of the fusion protein is shown in SEQ ID NO.2;

[0010] The MnCl2 solution was incubated with the cell membrane to obtain 2+ The initial product of the membrane component will be combined with Mn 2+ The initial membrane component product was added dropwise to the cell membrane component for secondary coating to obtain antibody-Mn 2+ Modified cell membranes;

[0011] Antibody-Mn 2+ The modified cell membrane was added to the liposome extruder, and the extrudate was collected and centrifuged to obtain the modified exosome αPDL1@Mn 2+ Then, DOX was loaded by electroporation to obtain antibody-metal modified exosomes αPDL1@DOX-Mn 2+ .

[0012] SEQ ID NO.1:

[0013] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANI

[0014] KQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGW

[0015] FGELAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGER

[0016] ATLSCRASQRVSSSYLAWYGGKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGGGTKVEIKRGGGGS.

[0017] SEQ ID NO.2:

[0018] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANI

[0019] KQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGW

[0020] FGELAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGER

[0021] ATLSCRASQRVSSSYLAWYGGKPGQAPRLLIYDASSRATGIPDRFSGSGSGTD

[0022] FTLTISRLEPEDFAVYYCQQYGSLPWTTFGGGTKVEIKRGGGGSGGGGSGGGGSGGGGSGGGGSAYLAVMVFALVLGWMNALYFTRGL.

[0023] The nanobody fragment containing the PDL1 targeting protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a V H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3;

[0024] The V H The amino acid sequence of CDR1 is shown in SEQ ID NO.3;

[0025] The V H The amino acid sequence of CDR2 is shown in SEQ ID NO.4;

[0026] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO.5;

[0027] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO.6;

[0028] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO.7;

[0029] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO.8.

[0030] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.9;

[0031] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10.

[0032] SEQ ID NO.3: GFTFSRY;

[0033] SEQ ID NO.4: KQDGSE;

[0034] SEQ ID NO.5: EGGWFGELAFDY;

[0035] SEQ ID NO.6:RASQRVSSSYLA;

[0036] SEQ ID NO.7: DASSRAT;

[0037] SEQ ID NO.8: QYGSLPWT.

[0038] SEQ ID NO.9:

[0039] EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANI

[0040] KQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

[0041] SEQ ID NO.10:

[0042] EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASS

[0043] RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK RGGGGS.

[0044] Furthermore, the membrane-anchored protein containing the nanobody fragment of the PDL1 targeting protein was transfected into HEK-293T cells, and after protein expression and extraction, the cell membrane transformed with the PDL1Nb-GFP-TRP PK1 fusion protein was extracted using a plasma membrane separation assay.

[0045] Further, the membrane-anchored protein containing the nanoantibody fragment of the PDL1 targeting protein was transfected into HEK-293T cells, and solution A and solution B were added in sequence, wherein solution A included 50 mM HEPES, 3 mM MgCl2 and 1 mM EGTA, and the pH was adjusted to 7.4; the solution B was 50 mM Tris-HCl, pH 7.4.

[0046] Furthermore, the MnCl2 solution was incubated with the cell membrane, placed on a shaker and repeatedly shaken and vortexed ultrasonically to obtain the MnCl2 solution. 2+The membrane component initial product, wherein the concentration of the MnCl2 solution is 15-25 mg / mL, the solute mass ratio of the MnCl2 solution to the cell membrane is 100-140:1, and the ultrasound is interval ultrasound.

[0047] Furthermore, antibody-Mn 2+ The modified cell membrane was added to the liposome extruder and extruded from large to small through a polycarbonate membrane with a particle size of 400nm-200nm-100nm. The extrudate was collected and centrifuged to obtain the modified exosome αPDL1@Mn 2+ .

[0048] Furthermore, the conditions for loading DOX are voltage 150-170 V, pulse time 80-120 ms, and interval 800-1200 ms.

[0049] Furthermore, after loading with DOX, the liquid mixture after electroporation was collected and filled with liquid and then centrifuged, and then washed again by centrifugation with PBS, and then resuspended in PBS and filtered through a 0.22 μm filter to obtain antibody-metal modified exosomes αPDL1@DOX-Mn 2+ .

[0050] The present invention also provides an antibody-metal modified exosome with anti-tumor immune enhancement effect, which is prepared by the above method.

[0051] The present invention also provides the use of the above-mentioned antibody-metal modified exosomes with anti-tumor immune enhancement effect in the preparation of anti-tumor drugs, wherein the anti-tumor drugs are used to kill tumors, induce ICD and / or promote cGAS-STING immune activation.

[0052] Furthermore, the anti-tumor drug is an intravenous injection solution.

[0053] A specific cell transmembrane peptide, wherein the transmembrane peptide is a peptide segment consisting of 24 amino acids in the fourth transmembrane region sequence (S4) of transient receptor potential vanilloid receptor 4 (TRPV4), and can be autonomously inserted into the phospholipid bilayer of the cell membrane;

[0054] The amino acid sequence of the S4 peptide segment is SEQ ID NO.11:

[0055] AYLAVMVFALVLGWMNALYFTRGL;

[0056] Beneficial Effects

[0057] In the present invention, Mn 2+The introduction of a purely biological exosome delivery system has innovated the construction form of related delivery carriers; the present invention uses exosome delivery for drug delivery to improve drug tumor penetration; the introduction of Mn 2+ It works synergistically with PDL1 Nb and DOX, and the trinity improves the efficacy and reduces systemic toxicity. The exosomes transformed by the method of the present invention have good functional effects of killing tumors, inducing ICD and activating cGAS-STING to promote immune activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 αPDL1@DOX-Mn 2+ Preparation and property characterization.

[0059] Figure 2 αPDL1@DOX-Mn 2+ The basis of action and functional effects.

[0060] Figure 3 Schematic diagram of the structure of the PCS2-Flag vector.

[0061] Figure 4 Mn 2+ Modification of exosomes αPDL1@DOX-Mn 2+ Preparation. (AB) Plasma membrane separation Western Blot detection and CLSM images of fusion protein cell localization; (C) Loading amount and loading efficiency curve of DOX loaded by electroporation method; (D) SEM-EDS analysis of Mn content of modified exosomes before and after electroporation loading; (E) Mn before and after electroporation 2+ ICP-MS quantification of loading.

[0062] Figure 5 αPDL1@DOX-Mn 2+ Parameter determination. (A) αPDL1@DOX-Mn 2+ TEM image of (B) αPDL1@DOX-Mn 2+ αPDL1@Mn 2+ , size distribution of exosomes; (C) zeta potential of three exosomes; (D) WB identification of three classic exosome markers; (E) αPDL1@DOX-Mn 2+ In vitro hemolysis test was used to detect the hemolysis rate; (F) αPDL1@DOX-Mn 2+ Particle size stability in 60% BSA environment; (G) αPDL1@DOX-Mn 2+ Compared with the yield of exosomes in the culture supernatant naturally secreted by cells.

[0063] Figure 6αPDL1@DOX-Mn 2+ Drug delivery behavior of Bank@Mn. (A) PDL1 expression in different tumor cell lines; (BC) Bank@Mn 2+ αPDL1@Mn 2+ Entry into different tumor cells and fluorescence quantification at 12 / 24h; (D) αPDL1@DOX-Mn 2+ The amount of DOX delivered into MDA-MB-231 cells at 1 / 4 / 12 h; (E) DiR-labeled αPDL1@DOX-Mn 2+ With Blank@DOX-Mn 2+ In vivo imaging of mice after tail vein injection.

[0064] Figure 7 αPDL1@DOX-Mn 2+ (AB) CRT expression detection; (C) ATP release detection; (D) ELISA detection of HMGB-1 release; (E) Introduction of Mn 2+ (FH) The toxic effects of different drugs on different tumor cells and normal human liver cells; (IJ) Flow cytometry images and statistical results of DC maturation level; (KL) Downstream T cell proliferation percentage and statistical results. DETAILED DESCRIPTION

[0065] (I) Experimental content

[0066] 1. Antibody-metal modification of exosomes αPDL1@DOX-Mn 2+ Preparation and characterization of

[0067] After preliminary exploration, αPDL1@DOX-Mn 2+ The preparation comprises the following steps:

[0068] (1) Transfecting a membrane-anchored protein containing a Nanobody (Nb) fragment of the PDL1 targeting protein PDL1 into HEK-293T cells, and extracting the cell membrane components modified by the fusion protein after successful protein expression;

[0069] (2) The MnCl2 solution was incubated with the extracted cell membrane components at room temperature, placed on a shaker and repeatedly shaken and vortexed with ultrasound, and then the MnCl2 solution was 2+ The initial membrane component product was added dropwise to the cell membrane component for secondary coating to obtain antibody-Mn 2+ Modified cell membrane components;

[0070] (3) The secondary coated product was added to a liposome extruder and extruded through a polycarbonate membrane with a particle size of 400 nm-200 nm-100 nm to form modified exosomes αPDL1@Mn 2+ Then, DOX was loaded by electroporation to prepare antibody-metal modified exosomes αPDL1@DOX-Mn 2+ .

[0071] After the preparation of αPDL1@DOX-Mn 2+ Afterwards, the exosome-related characterization indicators and application properties were determined:

[0072] (1) Quantitative detection of Mn in modified exosomes 2+ Load conditions, prove Mn 2+ The successful introduction of

[0073] (2) Based on the international consensus "MISEV 2018", the morphology, particle size distribution, protein markers, surface charge, etc. of the modified exosomes were studied by comparing them with the natural exosomes secreted by cells;

[0074] (3) To test and modify the basic properties of exosomes such as biocompatibility and circulation stability based on application requirements.

[0075] 2. Antibody-metal modification of exosomes αPDL1@DOX-Mn 2+ The basis of action and functional effects

[0076] Using αPDL1@DOX-Mn 2+ By co-incubating with PDL1-positive tumor cells in vitro, the modified exosomes were preliminarily confirmed to rely on the specific adsorption ability of PDL1 Nb through direct observation imaging and objective quantitative detection. At the same time, a mouse tumor model was constructed, and in vivo animal imaging was performed after administration via the tail vein to visualize the local accumulation of modified exosomes in the tumor and the organ distribution.

[0077] At the same time, αPDL1@DOX-Mn 2+ Tumor cells were treated to confirm their ability to directly kill tumors and induce apoptosis. Tumor cells were treated with drugs and co-cultured with DC cells, T cells and other immune cells in a Transwell system that interacted with each other materially to detect the phosphorylation level of downstream cGAS-STING pathway proteins, DC maturation and T cell proliferation level.

[0078] (II) Experimental methods

[0079] 1. Detection of the expression of PDL1 Nb-GFP-TRP PK1 fusion protein in cells

[0080] 1.1. Spatial localization imaging of PDL1 Nb-GFP-TRP PK1 in cells

[0081] The expression vector of PDL1 Nb-GFP-TRP PK1 fusion protein was constructed as follows: the sequence encoding αPDL1 Nb (as shown in SEQ ID NO.1) synthesized by GenScript and the sequence encoding TRP PK1 (as shown in SEQ ID NO.11) were fused into the PCS2-Flag vector linearized by double restriction enzyme digestion through homologous recombination ( Figure 3 ). Each functional area is connected through a flexible Linker (GS) n After conventional molecular cloning, amplification, and quantification, the Hieff The PDL1 Nb-GFP-TRP PK1 expression vector and the cell localization plasmids of the nucleus, mitochondria, peroxisomes and endoplasmic reticulum were co-transfected into HEK-293T cells that had been plated in 6-well plates for 24 hours in advance using liposome nucleic acid transfection reagent. The culture medium was replaced with complete medium 6 hours after transfection. The cells were digested and transferred to confocal glass bottom dishes 24 hours after transfection. BS-15-GJM) for 6 hours, remove the culture medium and wash once with PBS. Then, incubate with DiO solution (working concentration 10 μM) diluted with PBS at 37°C for 20 minutes to stain the cell membrane. After the complete culture medium is terminated, discard and wash. Fix with 4% paraformaldehyde at room temperature for 10 minutes, wash, and add DAPI solution (working concentration 10 μg / ml) for 5 minutes. Use TCS SP8 confocal laser scanning microscope (CLSM) from Leica, Germany, to visually image its spatial localization in the cell.

[0082] 1.2. Plasma membrane fractionation assay of PDL1 Nb-GFP-TRP PK1 distribution in cells

[0083] After the expression vector of PDL1 Nb-GFP-TRP PK1 fusion protein was transferred into HEK293T cells for 24 hours, the cells were digested and collected into centrifuge tubes, and washed 1-2 times with PBS. According to the amount of cells, an appropriate amount of solution A (HEPES 50mM, MgCl2 3mM and EGTA 1mM, pH adjusted to 7.4, per 2×10 6 ~5×10 6Add 1 mL of solution B per cell) and use a microsyringe to homogenize the cells by blowing them several times. Add an equal amount of solution B (50 mM Tris-HCl, pH 7.4) and centrifuge at 40,000g for 10 min at 4°C. Add an equal amount of homogenate from the previous step to the precipitate, homogenize the cells again, add Tris-HCl, and centrifuge under the same conditions for a total of three operations. Collect the precipitate and add an appropriate amount of Tris-HCl to resuspend it. Determine the protein sample concentration by BCA, add loading buffer, bathe at 95°C for 10 min, and detect by Western Blot.

[0084] 2. Antibody-metal modification of exosomes αPDL1@DOX-Mn 2+ Preparation and purification of

[0085] The cell membrane was extracted according to the plasma membrane separation test, and ddH20 was added for ultrasonic dispersion and BCA quantification to adjust the suspension to 1 mg / mL. 20 mg / mL MnCl2 solution was mixed with the cell membrane at a solute mass ratio of 1,200:10, and then treated with ultrasound at 4°C for 3-4 times (100-200W for 10s, with an interval of 10s), placed on a horizontal shaker for 10min, and repeated 4-5 times; then centrifuged at 12,000rpm for 30min at 4°C to collect the antibody-Mn 2+ Modified cell membrane components. ddH20 was ultrasonically dispersed, BCA was quantified and adjusted to a 2 mg / mL suspension, which was added dropwise to the 2 mg / mL cell membrane suspension, and ultrasonic vortexing was repeated until the solution was transparent and no precipitation appeared. The obtained solution was added to the liposome extruder, and polycarbonate membranes with pore sizes of 400nm-200nm-100nm were placed at the outlet for extrusion from large to small, and the extrudate was collected and centrifuged at 100,000g 4℃ for 90min using the American Beckman ultraspeed refrigerated centrifuge L-100XP to obtain αPDL1@Mn 2+ The exosome αPDL1@Mn was resuspended in EL electroporation buffer (Cat. No. H10305) produced by Suzhou ettabiotech. 2+ The company's X-Porator H1 electroporator was used to load DOX into αPDL1@Mn 2+ (voltage 160V, pulse time 100ms, interval 1000ms), collect the liquid mixture after electroporation, fill it up with liquid, centrifuge at 100,000g 4℃ for 90min, and then centrifuge again with PBS to wash, resuspend in a small volume of PBS and filter through a 0.22μm filter membrane to finally obtain the antibody-metal modified exosomes αPDL1@DOX-Mn after separation and purification. 2+ .

[0086] 3. Antibody-metal modification of exosomes αPDL1@DOX-Mn 2+ Characterization and identification

[0087] 3.1. Rough quantification of exosome production by BCA

[0088] The working solution was prepared using the BCA protein concentration assay kit (catalog number: HBCA-500) from hycezmbio at a ratio of solution A: solution B = 50:1. 200 μL was added to each well of a 96-well plate containing 10 μL of exosome suspension, incubated at 37°C for 30 min, and the absorbance at 562 nm was read using an enzyme reader. The sample concentration was calculated based on the standard curve.

[0089] 3.2. Modification of Mn in exosomes 2+ Determination of DOX content

[0090] First, the modified exosomes were treated with 1% Triton-X100 for 10 min, and then SEM-EDS analysis was performed using a ZeissSIGMA field emission scanning electron microscope from Zeiss, UK, to image and map the presence of Mn on the modified exosomes. An ICP-MS instrument (model: PQ-MS) from analytikjena, Germany, was used to analyze the Mn content in the modified exosomes. 2+ The content of DOX was accurately determined. After Triton treatment, the absorbance at 480 nm was measured using an ELISA reader and calculated based on the standard curve.

[0091] 3.3. Characterization of exosome-related properties of modified exosomes

[0092] TEM images were taken using a Hitachi HT7700 transmission electron microscope; the particle size and zeta potential were characterized using a Zetasizer Nano ZS nanoparticle size potentiometer from Malvern, UK; following the gold standard of "three positives and one negative" in MISEV 2018, Western Blot was used to detect the expression of CD9, CD63, HSP70, and Calnexin. (Proteintech, Cat No.60232-1-Ig, Cat No.25682-1-AP, Cat No.10995-1-AP, Cat No.10427-2-AP)

[0093] 3.4. Biocompatibility and circulation stability of modified exosomes

[0094] Mouse red blood cells were extracted and a 2% red blood cell suspension was prepared using physiological saline. 500 μL of different doses of αPDL1@DOX-Mn 2+Mix with 500 μL of red blood cell suspension, and use ddH2O and saline as negative and positive controls, and incubate at 37°C for 1 hour. Centrifuge at 2,400 rpm for 5 minutes, take the supernatant and measure the absorbance at 540 nm in an ELISA reader, and calculate the hemolysis rate according to the following formula:

[0095]

[0096] αPDL1@DOX-Mn 2+ The particles were placed in a 60% BSA blood simulation environment, and the particle size change was measured using a Zetasizer Nano ZS nanoparticle size potential analyzer produced by Malvern, UK.

[0097] 4. Modifying the drug delivery capacity of exosomes

[0098] Different cancer cell lines (A549, MDA-MB-231) were cultured at 3×10 5 After 24 h, αPDL1@Mn stained with DiI was plated into confocal glass bottom dishes at a density of 1 / well. 2+ After adding to the dish and incubating at 37°C for 12 / 24h, the cells were stained with DiO (working concentration 10μM) and DAPI (working concentration 10μg / ml) according to the above method, and confocal images of the three fluorescences were taken using CLSM; or the cells were initially plated into 6-well cell culture plates at the same density and αPDL1@DOX-Mn was added. 2+ After incubation for 1 / 4 / 12 h, cells were digested and collected, and the fluorescence signal generated by DOX in the PE channel was measured using a CytoFLEX S flow cytometer from Beckman, USA. 6 4T1-Luc cells were injected into the subcutaneous mammary fat pad of 6-week-old Balb / c female mice, and DiR-stained αPDL1@DOX-Mn was injected into the tail vein after tumor formation. 2+ With Blank@DOX-Mn 2+ The Xtreme B1 small animal in vivo imager from BRUKER, USA, was used to detect the local drug accumulation in the tumor.

[0099] 5. Modification of Tumor Killing and Apoptosis Induction Levels of Exosomes

[0100] MDA-MB-231 cells were cultured at a rate of 1×10 4 Each well was plated into a 96-well cell culture plate and the modified exosomes αPDL1@DOX-Mn were added 2+ After 24 hours of treatment, CCK8 kit was used Detect cell viability. At the same time, similar treatment can be performed in a 6-well cell culture plate. After digestion and collection of cells, the counts are adjusted to a density of 1×106 cells / mL, take 100 μL of cell suspension and use The cells were stained with the company's Annexin V-FITC / PI apoptosis kit and detected in the FITC / PE channel of the flow cytometer.

[0101] 6. ICD induction of modified exosomes

[0102] CRT exposure: MDA-MB-231 cells were cultured at 3×10 5 Each well was plated into a 6-well cell culture plate and the modified exosomes αPDL1@DOX-Mn were added 2+ After 24 h of treatment, the cells were collected and washed, counted, and resuspended in ice-cold PBS solution containing 10% FBS and 1% sodium azide to a density of 1×10 6 Cells / mL. Take 100μL of cell suspension and add 4% paraformaldehyde to a final concentration of 0.01% to 1% to fix for 10 minutes, add Triton to a final concentration of about 0.1%, and incubate at room temperature in the dark for 15 minutes. Then wash the cells with 2mL PBS (containing 0.1% triton), centrifuge at 300g for 5 minutes at 4℃, and discard the supernatant. Add 1:1000 diluted CRT primary antibody (proteintech, Cat No.10292-1-AP) and incubate at 4℃ on a shaker for 1 hour, then discard the supernatant at 400g×5min, add ice PBS to resuspend, and then centrifuge and wash at 400g×5min, repeat 3 times. Use 1:200 diluted CoraLite488 fluorescent secondary antibody (proteintech), use the diluent to resuspend the cells and incubate at 4℃ or room temperature in the dark for 1 hour. After washing 3 times, resuspend with PBS, and use the CytoFLEX S flow cytometer of Beckman Company in the United States to detect the fluorescence intensity in the FITC channel. At the same time, MDA-MB-231 cells can also be plated into confocal glass bottom dishes at the initial stage, stained according to the above steps, and observed under a confocal microscope.

[0103] HMGB-1 release: MDA-MB-231 cells were cultured at 3×10 5 Each well was plated into a 6-well cell culture plate and the modified exosomes αPDL1@DOX-Mn were added 2+ After 24 hours of treatment, the supernatant was collected and ultrafiltrated, and the human high mobility group protein B1 (HMGB-1) ELISA research kit (Cat. No.: JM-04778H2) of Jiangsu Jingmei Biotechnology Co., Ltd. was used to detect the content of HMGB-1 released by cells in the supernatant.

[0104] ATP release: MDA-MB-231 cells were cultured at a rate of 1×10 4 Each well was plated into a 96-well cell culture plate and the modified exosomes αPDL1@DOX-Mn were added2+ After 24 hours of treatment, the supernatant was collected and the samples were treated with the Meilun Bio-Enhanced ATP Detection Kit (Cat. No.: MA0440-1), and then the fluorescence quantification was performed using the TECAN SPARK multifunctional microplate reader.

[0105] 7. Modification of cGAS-STING activation and immune regulation function of exosomes

[0106] The Transwell model was used for the study. 4T1 cells were plated at 2 × 10 4 αPDL1@DOX-Mn 2+ After 24 h of treatment, the lower chamber was first added with pre-extracted mouse BMDCs (10 ng / ml IL-4 and 20 ng / ml GM-CSF to induce mouse bone marrow cells in vitro for 8 days, and half of the medium was replaced every 2 days. The density of the lower chamber was 1×10 5 After 24 hours of co-incubation with 4T1 cells pretreated with drugs, BMDCs were collected from the lower chamber and the cells were lysed to extract proteins and then Western Blot was used to detect the phosphorylation level of STING. At the same time, CD86-APC / CD80-FITC staining (Proteintech) was used to detect DC maturation by flow cytometry. Finally, after BMDCs were co-incubated with 4T1 cells pretreated with drugs in the lower chamber for 24 hours, mouse spleen cells stained with CFSE (Yisheng Bio Cell Proliferation and Tracing Detection Kit, Cat. No.: 40714ES76) were added at 4×10 5 The cells were added to the lower layer of the chamber at a density of 100 cells / well and incubated for 24 hours. After staining with CD3-APC (Elabscience), T cell proliferation was detected by flow cytometry.

[0107] (III) Experimental results

[0108] 1. Preparation and characterization of antibody-metal modified exosomes αPDL1@DOX-Mn 2+

[0109] A targeting protein containing PDL1 Nb has been constructed and confirmed to be widely expressed on the membrane structure of HEK-293T cells ( Figure 4 AB), to prepare functional cell membrane, Mn 2+ The efficiency of DOX loading by electroporation is about 20% ( Figure 4 C), but due to the direct addition of Mn 2+ This will change the dielectric properties of the buffer system and affect the normal electroporation process, so other methods of loading Mn should be considered. 2+ .

[0110] Mn 2+It can form complexes with various proteins, such as ferritin-iron ion system. Since the modified cell membrane contains a large amount of protein, it can be used to adsorb Mn 2+ Exosomes were then prepared. It has been confirmed that Mn 2+ It can bind to the cell membrane and after a series of preparation processes, the final product αPDL1@DOX-Mn 2+ Mn accumulation was detected in Figure 4 DE).

[0111] 2. Determination of Antibody-Metal Modified Exosomes αPDL1@DOX-Mn 2+ The property parameters

[0112] Since the traditional mainstream method for preparing exosomes is to extract the supernatant of cell culture medium and perform ultracentrifugation to separate and purify the exosomes actively secreted by cells, which is different from the passive method we used to first prepare the modified membrane components and then extrude them through the membrane to form exosomes, after preparing the modified exosomes, we tested its basic properties such as exosome-related protein markers, particle size distribution, as well as important parameters such as biocompatibility and cyclic stability, and compared them with the properties of natural exosomes extracted by traditional methods. 2+ In the typical cup-shaped structure ( Figure 5 A), has a more uniform particle size distribution compared to natural exosomes, indicating that the homogeneity of the product is superior to that of natural exosomes ( Figure 5 B). αPDL1@DOX-Mn 2+ Compared with the natural ingredients, the zeta potential is more negative, indicating better stability ( Figure 5 C). In addition, compared with natural exosomes, which do not contain cytoplasmic proteins, endoplasmic reticulum-related proteins are positive in terms of protein markers, which is consistent with the difference in the formation process of the two exosomes ( Figure 5 D). In terms of application parameters, the modified exosomes have good biocompatibility, low hemolysis rate, and maintain stable particle size in a blood simulation environment ( Figure 5 EF). Moreover, the modified exosomes were prepared by membrane extrusion, which had a significantly higher yield than natural exosomes ( Figure 5 G), which is the basis for large-scale preparation.

[0113] In summary, modified exosomes are superior to natural exosomes in terms of stability, yield and uniformity of properties. This may be because our method of extruding the cell membrane through a polycarbonate membrane with a specific pore size improves the overall yield and ensures the uniformity of properties by maximizing the utilization of cell membrane components. At the same time, since only cell membrane components are used for exosome preparation, all cell contents are excluded, their stability in conventional storage systems is also improved.

[0114] 3. Confirmation of Antibody-Metal Modified Exosomes αPDL1@DOX-Mn 2+ Selective drug delivery behavior

[0115] The in vitro specific adsorption and drug delivery effects of modified exosomes on PDL1-positive cells based on PDL1 Nb were tested, which confirmed that it can continuously deliver DOX to PDL1-positive tumor cells ( Figure 6 D).

[0116] In a more complex in vivo environment, modified exosomes can accumulate locally in tumors through passive targeting of enhanced permeability and retention effect and active targeting of PDL1 Nb molecules. In vivo distribution studies after intravenous administration preliminarily confirmed their intratumoral accumulation ( Figure 6 E).

[0117] 4. Confirmation of Antibody-Metal Modified Exosomes αPDL1@DOX-Mn 2+ The effect of

[0118] ICD-induced release of DAMPs to activate cGAS-STING is the initiation link of anti-tumor immunity. We confirmed that the levels of calreticulin (CRT) on the surface of tumor cells and ATP / HMGB-1 in the culture supernatant increased significantly after drug treatment ( Figure 7 AD). And when introducing Mn 2+ After αPDL1@DOX-Mn 2+ Showing good ICD induction and cGAS-STING activation levels ( Figure 7 E), ultimately achieving good killing of tumor cells and protecting normal cells ( Figure 7 FH).

[0119] Downstream DC maturation and T cell activation levels were studied in a Transwell system in which BMDCs were co-cultured with drug-pretreated tumor cells. It was found that αPDL1@DOX-Mn 2+ Can significantly enhance DC maturation (CD80 + / CD86 + )percentage( Figure 7 I, J), and after adding mouse primary spleen cells pre-stained with CFSE, obvious T cell proliferation was observed ( Figure 7 K, L), indicating that αPDL1@DOX-Mn 2+ It can have an immune activation effect in vitro, preliminarily proving that αPDL1@DOX-Mn 2+ Has functional effectiveness.

Claims

1. A method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect, characterized in that: The following steps are involved: The membrane anchored protein of the nanobody fragment containing the PDL1 targeting protein is transfected into HEK-293T cells, and after the protein is expressed, the cell membrane transformed with the PDL1 Nb-GFP-TRP PK1 fusion protein is extracted; the amino acid sequence of the nanobody fragment containing the PDL1 targeting protein is shown in SEQ ID NO.1; the amino acid sequence of the fusion protein is shown in SEQ ID NO.2; The MnCl2 solution was incubated with the cell membrane to obtain 2+ The initial product of the membrane component will be combined with Mn 2+ The initial membrane component product was added dropwise to the cell membrane component for secondary coating to obtain antibody-Mn 2+ Modified cell membranes; Antibody-Mn 2+ The modified cell membrane was added to the liposome extruder, and the extrudate was collected and centrifuged to obtain the modified exosome αPDL1@Mn 2+ Then, DOX was loaded by electroporation to obtain antibody-metal modified exosomes αPDL1@DOX-Mn 2+ .

2. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: The membrane-anchored protein containing the nanobody fragment of the PDL1 targeting protein was transfected into HEK-293T cells, and after protein expression and extraction, the cell membrane transformed with the PDL1 Nb-GFP-TRP PK1 fusion protein was extracted using a plasma membrane fractionation assay.

3. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: The membrane-anchored protein containing the nanoantibody fragment of the PDL1 targeting protein was transfected into HEK-293T cells, and solution A and solution B were added in sequence. The solution A included 50 mM HEPES, 3 mM MgCl2 and 1 mM EGTA, and the pH was adjusted to 7.4; the solution B was 50 mM Tris-HCl, pH 7.

4.

4. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: The MnCl2 solution was incubated with the cell membrane, placed on a shaker and repeatedly shaken and vortexed with ultrasound to obtain the Mn-bound 2+ The membrane component initial product, wherein the concentration of the MnCl2 solution is 15-25 mg / mL, the solute mass ratio of the MnCl2 solution to the cell membrane is 100-140:1, and the ultrasound is interval ultrasound.

5. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: Antibody-Mn 2+ The modified cell membrane was added to the liposome extruder and extruded from large to small through a polycarbonate membrane with a particle size of 400nm-200nm-100nm. The extrudate was collected and centrifuged to obtain the modified exosome αPDL1@Mn 2+ .

6. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: The conditions for loading DOX were voltage 150-170 V, pulse time 80-120 ms, and interval 800-1200 ms.

7. The method for preparing antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 1, characterized in that: After loading with DOX, the liquid mixture after electroporation was collected and filled with liquid and then centrifuged. Then, it was washed by centrifugation again with PBS. After resuspending in PBS, it was filtered through a 0.22 μm filter membrane to obtain antibody-metal modified exosomes αPDL1@DOX-Mn 2+ . 8.An antibody-metal modified exosome with anti-tumor immune enhancement effect, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. The use of the antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 8 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is used to kill tumors, induce ICD and / or promote cGAS-STING immune activation.

10. The use of the antibody-metal modified exosomes with anti-tumor immune enhancement effect according to claim 8 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is an intravenous injection solution.

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