Construction of engineered extracellular vesicle of targeted vascular endothelial cell P-selectin and application of engineered extracellular vesicle in cancer treatment

Abstract

The invention relates to construction of engineered extracellular vesicles targeting a vascular endothelial cell P-selectin and application of the engineered extracellular vesicles in cancer treatment, and compared with natural extracellular vesicles, the engineered extracellular vesicles have the following characteristics: 1, the engineered extracellular vesicles can specifically target tumor cells and are harmless to normal tissue cells; anti-tumor nucleic acid drugs such as small interfering RNA of targeted EGFR and EGFR genes for inhibiting high expression of tumor cells can be carried, so that proliferation of the tumor cells is inhibited, and the anti-tumor effect is achieved. 3, the PSGL-1 over-expressed on the surface of the engineered extracellular vesicle membrane can specifically target P-selectin in tumor cells, so that the delivery efficiency and the delivery accuracy of the extracellular vesicle as a drug delivery carrier are greatly improved, and clinical transformation of the engineered extracellular vesicle for cancer targeted therapy is expected to be further enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the construction of engineered extracellular vesicles targeting vascular endothelial cell P-selectin, and the application of the method in treating various cancer models. Background Art

[0002] Cancer is a major public health problem worldwide and is considered the leading cause of death for the global population. Due to high recurrence and mortality rates, drug resistance, metastatic ability and poor prognosis, they have posed a serious threat to human health. Despite the great progress in medical technology over the years, the clinical data results of tumor treatment are not ideal. At present, the main means of treating tumors are radiotherapy and chemotherapy, but there are defects such as high toxicity, low delivery efficiency, tissue resistance, and insufficient drug targeting ability. The emergence of new targeted drugs has changed the tumor treatment model. Targeted therapy achieves the purpose of treating tumors by interfering with the growth, division and spread of cancer cells. Most targeted drugs are small molecule drugs or monoclonal antibody drugs, such as erlotinib or gefitinib, two signal transduction inhibitors targeting EGFR, and bevacizumab, an inhibitor targeting angiogenesis. However, this treatment method is slow to take effect, prone to targeted mutations and damage to biological barriers, and may cause tumor cells to develop resistance to targeted drugs. Another is gene therapy. In theory, this treatment method can target any pathogenic gene, such as using small interfering RNA as a gene therapy method, but the delivery vector is a barrier that limits its development. Especially in the treatment of central nervous system diseases such as brain tumors and neurodegenerative diseases, many drugs cannot effectively penetrate the blood-brain barrier and enter the brain due to the existence of the blood-brain barrier. Therefore, it is necessary to develop a more accurate, efficient and safe tumor drug targeting system.

[0003] Extracellular vesicles are a type of natural nanoparticles with a size of 30-1000nm. Almost all cells can release extracellular vesicles. It is widely present in body fluids such as saliva, blood, urine, and milk. These include microvesicles (100-1000nm), exosomes (50-150nm), apoptotic bodies (50nm-2μm), and tumor vesicles (1-10μm). Extracellular vesicles can carry proteins, lipids, and nucleic acids from donor cells to surrounding or distal recipient cells, thereby mediating intercellular communication. Because extracellular vesicles are secreted by natural cells and have the characteristics of low immunogenicity and low toxicity, extracellular vesicles can be used as a new type of biological delivery carrier.

[0004] A large number of studies have shown that extracellular vesicles can transport anti-tumor drugs for tumor treatment. In the field of nanomedicine, extracellular vesicles have been designed as therapeutic delivery carriers for a variety of anti-cancer drugs. Compared with commercial liposomes and polymer nanoparticles, the abundant membrane proteins on the surface of extracellular vesicles provide a very convenient modification platform. Through simple cell engineering methods, extracellular vesicles can carry a variety of functional membrane proteins. This modification of targeted proteins on the surface of extracellular vesicle membranes using genetic engineering methods can effectively improve the delivery accuracy of extracellular vesicles, mediate the targeting of gene therapy, and greatly improve the overall therapeutic effect.

[0005] P-selectin is a cell adhesion protein that is constitutively expressed in endothelial cells. Under endogenous cytokines or exogenous stimulation (such as ionizing radiation), endothelial cells are activated, and P-selectin translocates to the cell membrane and enters the vascular cavity. The membrane expression characteristics of P-selectin after endothelial cell activation are closely related to the occurrence and development of tumors. Studies have reported that P-selectin is highly expressed in a variety of tumor cells and tumor vasculature, but is almost not expressed in normal tissues. Therefore, P-selectin can be used as a target for targeting tumors and tumor microenvironment. There are currently two known ligands for P-selectin: the first is P-selectin glycoprotein ligand 1 (PSGL-1) expressed on various leukocytes, and the second is a 120kDa ligand expressed only on bone marrow cells.

[0006] Therefore, our research aims to construct an engineered extracellular microvesicle that can overexpress PSGL-1, and use the highly specific binding characteristics of PSGL-1 and P-selectin ligand receptor to efficiently and accurately deliver tumor therapeutic drugs to the tumor site. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for constructing an extracellular vesicle delivery vector that can specifically target tumor sites. The present invention greatly improves the delivery efficiency and accuracy of anti-tumor drugs by overexpressing P-selectin's ligand PSGL-1 on the surface of the extracellular vesicle membrane.

[0008] The invention includes an engineered extracellular vesicle targeting vascular endothelial cell P-selectin, wherein the extracellular vesicle is coated with an anti-tumor nucleic acid drug.

[0009] The present invention is achieved through the following technical solutions:

[0010] The first inventive point of the present invention is to synthesize three highly efficient overexpression PSGL-1 gene loops based on the design principle of synthetic biology, wherein the plasmid vector used is pcDNA6.2, and the promoter used is CMV promoter. The three plasmids are specifically:

[0011] One is a plasmid that can directly overexpress PSGL-1;

[0012] The second is a plasmid containing PSGL-1 fused to the extracellular vesicle membrane protein CD63 sequence;

[0013] The third is a plasmid that fuses PSGL-1 with the extracellular vesicle membrane protein LAMP2B sequence.

[0014] The second inventive point of the present invention is to specifically modify the extracellular vesicle membrane protein based on the design principle of synthetic biology so that the extracellular vesicle can specifically target different tissue sites.

[0015] The third inventive point of the present invention is to construct engineered extracellular vesicles that overexpress the P-selectin ligand PSGL-1 by utilizing the characteristic of ligand-receptor specific binding, targeting the high expression of P-selectin in tumor sites and the almost non-expression of this characteristic in normal tissues.

[0016] The fourth inventive point of the present invention is to provide the application of the above-mentioned engineered extracellular vesicles in tumor treatment, and to apply the engineered extracellular vesicles containing anti-tumor nucleic acid drugs to the treatment of different types of tumors.

[0017] The beneficial effects of the present invention are: based on the high expression of P-selectin in tumor sites and the almost non-expression of this characteristic in normal tissues, the extracellular vesicle membrane protein is specifically modified by means of synthetic biology, and anti-tumor nucleic acid drugs are carried to treat mice with various cancer types. This engineered extracellular vesicle not only has the ability to specifically target tumor cells, but also further improves the delivery efficiency of anti-tumor drugs, providing new treatment strategies and application prospects for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The plasmid maps are of the PSGL-1 (A), C63-PSGL-1 (B), and LAMP2b-PSGL-1 (C) overexpression plasmids of the present invention.

[0019] Figure 2 This is a schematic diagram of the engineered extracellular vesicle membrane structure of the present invention.

[0020] Figure 3 The protein and mRNA expression levels of PSGL-1 after infection of the overexpression plasmid in human renal epithelial cells of the present invention.

[0021] Figure 4 It is the expression level of the engineered extracellular vesicle membrane protein PSGL-1 of the present invention.

[0022] Figure 5 It is the targeted tumor tissue delivery efficiency of the engineered extracellular vesicles of the present invention; DAPI indicates the cell nucleus, PKH26 indicates the engineered extracellular vesicles, and GFP indicates the tumor cells.

[0023] Figure 6 The mRNA expression level of EGFR in tumor tissues (A: glioblastoma, B: lung cancer) after the engineered extracellular vesicles of the present invention carry anti-tumor nucleic acid drugs (small interfering RNA).

[0024] Figure 7 The efficiency of the engineered extracellular vesicles of the present invention in targeting non-tumor tissues (A: heart, B: kidney, C: lung, D: spleen). DETAILED DESCRIPTION

[0025] The application effects of the present invention are elaborated in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention. However, it should be noted that the present invention is not limited to these embodiments.

[0026] Example 1 Construction of PSGL-1, CD63-PSGL-1 and lamp2b-PSGL-1 plasmids

[0027] We used restriction endonucleases to double-digest the PCDNA6.2 plasmid vector, ran 1% agarose gel and recovered larger gene fragments, synthesized PSGL-1-siEGFR, CD63-PSGL-1-siEGFR and lamp2b-PSGL-1-siEGFR fragments, double-digested the double-stranded PSGL-1-siEGFR, CD63-PSGL-1-siEGFR and lamp2b-PSGL-1-siEGFR sequences, recovered the digestion products through a column, and used T4 ligase to connect the vector and the insert fragment that had also been double-digested; the resulting ligation products were subjected to transformation experiments and spread on resistant plates; single clones were picked on the next day, sequencing confirmed the correctness of the plasmid sequence, and positive colonies with successful cloning were screened out.

[0028] Specific experimental steps:

[0029] 1. Enzyme Digestion

[0030] The plasmid pcDNA6.2 was double-digested with BamHI and XhoI, and the reaction system was as follows: 37°C for 30 min; 85°C for 5 min (different enzymes have different inactivation temperatures). After the digestion reaction was completed, the product was run on gel electrophoresis, and the target fragment was recovered from the gel.

[0031] Enzyme digestion system (20 μL)

[0032]

[0033] 2. Enzyme Linking

[0034] The double-stranded DNA was connected to the vector pcDNA6.2. The connection system was:

[0035] Ligation system (10 μL)

[0036]

[0037]

[0038] 16℃ overnight

[0039] 3. Perform conversion sequencing

[0040] The sequencing primer was the universal primer CMV-F.

[0041] Example 2 Construction of overexpression cells and verification of extracellular vesicles

[0042] The construction of overexpression cells is divided into two parts. The first step is to use human renal epithelial cells to prepare lentivirus containing PSGL-1 / CD63-PSGL-1 / lamp2b-PSGL-1 genes to obtain lentiviral suspension. The second step is to infect new human renal epithelial cells with the obtained virus suspension, select stable monoclonal cell lines after drug screening, collect extracellular vesicles of stable cell lines and detect the expression efficiency of PSGL-1.

[0043] Specific experimental steps:

[0044] 1. Lentiviral packaging

[0045] A. Subculture human renal epithelial cells and plate the cells in a 100 mm dish at a ratio of 1:3. Transfect when the cell density reaches about 70%.

[0046] B. The target plasmid was mixed with psPAX2 and pMD2G at a molar ratio of 10:5:1, and then transfection was performed according to the instructions of lipofectamine 2000.

[0047] Lentivirus infection reagent ratio

[0048]

[0049] C. After 6 h, the medium was replaced with fresh medium containing 2% FBS.

[0050] D. After 48 h, the lentiviral particles were collected and filtered using a 0.22 μm filter.

[0051] E. Store the lentiviral stock solution in a -80°C refrigerator.

[0052] 2. Lentivirus infection

[0053] A. For cell passaging, cells were plated into 6-well plates at a ratio of 1:3.

[0054] B. After the cells adhered for 12-18 hours, the cell density was 30-40%, and the volume of lentivirus required for the lentivirus titer and MOI was calculated based on the cell number.

[0055] C. Inactivate fetal bovine serum at 56°C for 30 min. Discard the original culture medium in the 6-well plate, wash the cells with PBS, and replace with culture medium containing 2% inactivated FBS.

[0056] D. Thaw the lentivirus on ice, add the lentivirus and polybrene (final concentration 4 μg / mL) to the 6-well plate according to the calculated volume, shake gently to mix, and place in a 37°C cell culture incubator for continued culture.

[0057] E. After 24 hours, discard the virus infection solution, replace with new 2% FBS culture solution, add puromycin, shake well, and place in a 37℃ cell culture incubator for continued culture.

[0058] F. Replace the culture medium containing puromycin every 24 hours. If the cell density exceeds 90% during the screening period, normal subculture is performed, and the new subculture cells are allowed to adhere to the wall for 12 hours before continuing the puromycin screening.

[0059] G. The cell line after one week of screening is the stably infected cell line, and drug screening and culture will continue.

[0060] 3. Monoclonal cell line screening

[0061] A. After the target cells have grown to confluence, digest and resuspend them normally.

[0062] B. Take an appropriate amount of resuspended cell solution for dilution and cell counting. To ensure 1.5 cells per well, take a small amount of resuspended solution containing 450 (1.5×60×5=450) cells, dilute to 90 ml (1.5×200×60×5=90 ml) with puromycin culture solution and mix thoroughly. Spread this dilution on 5 96-well plates, spread 200ul / well of cell suspension on the middle 60 wells of each 96-well plate, and block the remaining wells with 200ul / well PBS.

[0063] C. After plating, observe whether the medium needs to be changed every two days. After culturing for two weeks, observe the number of cells in each well of the culture plate under an inverted microscope and mark the wells with 1-2 cells growing.

[0064] D. Expand the labeled positive cells step by step from 96-well plate to 48-well plate to 12-well plate until the required cell amount is obtained for monoclonal cell line verification.

[0065] E. Verification of monoclonal cell lines

[0066] 4. Extracellular Vesicle Immunoprecipitation

[0067] A. Lyse cells and extracellular vesicles with lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM PMSF, 1% Protease Inhibitor Cocktail and 10 U / mL RNase inhibitor) on ice for 30 min.

[0068] B. The lysate was centrifuged at 16,000×g for 10 min at 4°C.

[0069] C. Pipette the supernatant into a new EP tube and divide it into two groups. Add anti-Flag antibody (2-3 μg) or normal IgG and incubate on a rocker at 4°C overnight.

[0070] D. On the next day, 40 μL of Protein A / G PLUS-Agarose beads were added to each tube, and then the mixture was incubated on a rocker at 4°C for 2 h.

[0071] E. Collect the immunoprecipitate after centrifugation at 2,500 rpm for 5 min at 4°C and discard the supernatant.

[0072] F. Centrifuge at 2,000 rpm for 3 min at 4°C with 1 mL of lysis buffer and wash four times.

[0073] G. After elution, beads were collected, proteins were lysed, and Western blot analysis was performed.

[0074] Result analysis: According to the attached Figure 3 , 4 It can be confirmed that the stable cell line overexpressing PSGL-1 was successfully constructed, and the overexpressed PSGL-1 protein could also be detected in the extracellular vesicles secreted by them.

[0075] Example 3 In vivo delivery efficiency of engineered extracellular vesicles

[0076] The engineered extracellular vesicles stained with PKH26 were injected into mice through the tail vein. Four hours later, the brain, heart, lung, kidney and spleen of the mice were collected to detect the targeting status of the engineered extracellular vesicles in the mice.

[0077] Specific experimental steps:

[0078] 1. Immunofluorescence

[0079] A. Fluorescently labeled extracellular vesicles were injected into mice via the tail vein, and the mice were anesthetized 4 hours later.

[0080] B. Perfusion preparation: Before perfusion, prepare PBS, scalp needle, 10 mL syringe and 4% paraformaldehyde. Connect the scalp needle to the 10 mL syringe, draw PBS and expel bubbles. Use another 10 mL syringe to draw 4% paraformaldehyde and expel bubbles.

[0081] C. Spray 75% alcohol on the surface of the mouse, open the abdominal cavity and chest cavity of the mouse to expose the liver and heart, cut the right atrial appendage with scissors, insert a scalp needle into the left ventricle, push the syringe to make PBS flow out from the right atrial appendage until the liver turns from red to whitish, replace the syringe containing PBS with a syringe containing 4% paraformaldehyde, and continue injecting until the tissue hardens;

[0082] D. Fixation: All tissues of the mouse were fixed in 4% paraformaldehyde for about 10 hours;

[0083] E. Dehydration: Transfer the tissue to a 20% sucrose solution for dehydration. After sinking to the bottom, continue to transfer the tissue to a 30% sucrose solution for further dehydration. The gradient dehydration time should not exceed 24 hours.

[0084] F. Embedding: After dehydration, place the tissue in a folded mold, add OCT to cover the tissue, and place in a -20℃ refrigerator to cool down;

[0085] G. Sectioning: Take the embedded tissue out of the mold, apply OCT to connect it to the stage, cool it down on the quick-freeze table to make the tissue and the stage form a whole, fix it on the freezing head, slice it at a thickness of 8 μm, stick it to the center of the slide, and dry it in the dark;

[0086] H. Staining:

[0087] a. Circle the slice position with an immunohistochemistry pen and dry the slices away from light at room temperature;

[0088] b. Slowly wash the OCT attached to the slices with PBS for 3 times, 3 minutes each time;

[0089] c. Block with blocking solution at room temperature in the dark for 1 hour. The blocking solution is PBS solution with TritonX-100 and 5% BSA added.

[0090] d. Prepare the primary antibody with blocking solution, dilute the antibody according to the instructions, incubate at room temperature in the dark for 3 hours, or incubate in a 4°C refrigerator overnight;

[0091] e. After incubation, discard the antibody and wash with PBS three times, 3 minutes each time;

[0092] f. Prepare fluorescent secondary antibody with blocking solution, dilute the antibody according to the instructions, and incubate at room temperature in the dark for 1 hour;

[0093] g. After incubation, discard the antibody and wash with PBS 3 times, 3 minutes each time;

[0094] h. Dilute DAPI at a ratio of 1:1000, add it dropwise to the tissue surface, and incubate at room temperature in the dark for 20 minutes;

[0095] i. After the incubation, discard the liquid and wash with PBS three times, each time for 3 minutes;

[0096] j. Add anti-fluorescence quenching agent to the tissue surface, cover with a coverslip, and take pictures.

[0097] Result analysis: Figure 4 , 7 The results showed that engineered extracellular vesicles overexpressing PSGL-1 could be more efficiently and accurately delivered to tumor tissue sites compared with ordinary extracellular vesicles.

[0098] Example 4 Verification of the in vivo therapeutic effect of engineered extracellular vesicles

[0099] The engineered extracellular vesicles carrying siEGFR were collected and injected intravenously into the glioblastoma and lung cancer model mice. 1×10 10 particles / day, once a day for a week. After the treatment, the mouse tumor tissues were taken to detect the expression level of EGFR in the tumor cells to evaluate the treatment effect.

[0100] Result analysis: as attached Figure 6 As shown, the anti-tumor nucleic acid drug siRNA carried by engineered extracellular vesicles can effectively reach the tumor site and reduce the expression level of tumor epidermal growth factor EGFR.

[0101] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An engineered extracellular vesicle targeting vascular endothelial cell P-selectin, characterized in that: The engineered extracellular vesicles are extracellular vesicles derived from human renal epithelial cells transfected with a plasmid that overexpresses PSGL-1, the exosome membrane surface has overexpressed PSGL-1 protein, and the extracellular vesicles carry anti-tumor nucleic acid drugs; the anti-tumor nucleic acid drug is siRNA.

2. The engineered extracellular vesicle targeting vascular endothelial cell P-selectin according to claim 1, characterized in that: There are three ways to overexpress PSGL-1 protein: direct overexpression of PSGL-1, overexpression of PSGL-1 by fusion with LAMP2b, and overexpression of PSGL-1 by fusion with CD63. The anti-tumor nucleic acid drug is siRNA targeting the EGFR gene.

3. The method for preparing an engineered extracellular vesicle targeting vascular endothelial cell P-selectin according to claim 1 or 2, characterized in that: The steps include: Three plasmids for overexpressing PSGL-1 were constructed; The three plasmids overexpressing PSGL-1 and the lentiviral helper plasmid were co-transfected into human renal epithelial cells respectively, and single cells successfully infected were screened and cultured to obtain monoclonal cell lines with high expression of PSGL-1 protein; The cells are cultured and the engineered extracellular vesicles are collected.

4. A method for preparing an engineered extracellular vesicle targeting P-selectin in vascular endothelial cells as claimed in claim 3, characterized in that: The culturing cells and collecting engineered extracellular vesicles specifically include: Monoclonal cell lines that highly express PSGL-1 protein are cultured on a large scale, and the supernatant is collected and separated and extracted by ultracentrifugation to obtain engineered extracellular vesicles loaded with PSGL-1 protein and targeted to tumor tissues.

5. A construction of an engineered extracellular vesicle targeting vascular endothelial cell P-selectin as claimed in claim 1 or 2 and its application in cancer treatment, wherein the tumor is glioblastoma and lung cancer.

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