Engineered exosome drug delivery system for over-expression of CD24 and preparation method of engineered exosome drug delivery system

Abstract

The invention provides an engineering exosome drug delivery system for over-expression of CD24 and a preparation method thereof, and belongs to the technical field of gene engineering. CD24 is overexpressed on a membrane of the engineered exosome, and a gene sequence of a plasmid of the overexpressed CD24 is as shown in SEQ ID No. 1; a CD24 sequence is fused to the N end of an I-type transmembrane protein structure LAMP2B on a membrane of the engineered exosome, a glycosylation sequence GNSTM is introduced to the N end of the fusion sequence, and an exosome binding peptide Signal peptide fragment is fused to the N end of the glycosylation sequence GNSTM. The engineering exosome for over-expressing CD24 on an exosome membrane is successfully constructed by using a fusion gene for coding transmembrane proteins LAMP2B and CD24, the aggregation and protein adsorption phenomena of the engineering exosome can be effectively retarded, the drug delivery targeting of the engineering exosome is enhanced, the in-vivo duration of the engineering exosome is prolonged, and the delivery performance of the engineering exosome is further improved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to an engineered exosome drug delivery system overexpressing CD24 and a preparation method thereof. Background Art

[0002] Exosomes (Exosomes, exo), as a natural biological carrier, are tiny vesicles with a diameter of 30 nm - 150 nm secreted by cells. They have good biocompatibility, excellent biological barrier penetration ability, low immunogenicity, and rich cell sources. Therefore, they show significant advantages in in vivo drug delivery and have important application values in the fields of biology and medicine. However, the natural characteristics of exosomes and their rapid clearance mechanism in vivo limit the efficiency of their drug delivery, and it is urgent to develop a more efficient exosome drug delivery carrier. Summary of the Invention

[0003] The purpose of the present invention is to provide an engineered exosome drug delivery system overexpressing CD24 and a preparation method thereof to solve at least one of the technical problems existing in the above background art.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, the present invention provides an engineered exosome drug delivery system overexpressing CD24. CD24 is overexpressed on the membrane of the engineered exosome, and the gene sequence of the plasmid overexpressing CD24 is as shown in SEQ ID No. 1.

[0005] As a further limitation of the first aspect of the present invention, the CD24 sequence is fused to the N-terminus of the type I transmembrane protein structure LAMP2B on the membrane of the engineered exosome. A glycosylation sequence GNSTM is introduced at the N-terminus of the fusion sequence, and a Signal peptide fragment is fused to the N-terminus of GNSTM. The glycosylation sequence GNSTM, that is, the GNSTM motif, is a protein motif located on the surface of extracellular vesicles (such as exosomes) and has specific amino acid sequence characteristics. The GNSTM motif is usually related to the glycosylation sites on the surface of exosomes, and these sites can be fused and expressed with the exosome marker CD63, the luminal protein Hspa8, and the extracellular glycosylation site GNSTM, thereby affecting the biophysical properties of exosomes. The Signal Peptide fragment, that is, the signal peptide, is a short peptide sequence of 15 - 30 amino acids located at the N-terminus of a protein. It is mainly responsible for guiding the newly synthesized protein to be transported to a specific cell region (such as the cell periplasm). Through its hydrophobic structure and specific sequence characteristics, it interacts with the intracellular transport system and is finally excised by an enzyme after the transport is completed to ensure the correct folding and localization of the protein. As a further limitation of the first aspect of the present invention, the plasmid overexpressing CD24 includes adding a GNSTM motif to the N-terminus of the Myc fragment in the Myc-CD24 fragment, fusing the Signal peptide fragment to the N-terminus of the GNSTM motif, and fusing the LAMP2B fragment to the C-terminus of the CD24 fragment in the Myc-CD24 fragment; wherein, the Signal peptide fragment and the LAMP2B fragment are cloned from the pcDNA GNSTM-3-Flag-10-Lamp2b-HA plasmid by PCR technology, and the Myc-CD24 fragment is cloned from the pCMV-Cd24-FLAG-Neo plasmid. Among them, the pcDNA GNSTM-3-Flag-10-Lamp2b-HA plasmid is provided by Shanghai Kelei Biotechnology Co., Ltd.; the pCMV-Cd24a-FLAG-Neo plasmid is provided by Wuhan Miaoling Biotechnology Co., Ltd.

[0006] In a second aspect, the present invention provides a method for preparing the engineered exosome drug delivery system overexpressing CD24 as described in the first aspect. The plasmid overexpressing CD24 is mixed evenly with Opti-MEM, psPAX2, pMD2.G, and PEI transfection reagent and then added to a well plate to obtain cell supernatant, which is used to infect HEK293T cells. The cells are transferred to a 10 cm dish for normal culture, and the culture supernatant of the cells is collected. The cells and cell debris are removed by centrifugation, and the filtered supernatant is centrifuged to granulate the exosomes, and the supernatant is discarded; the exosome particles are resuspended in DPBS and ultracentrifuged to remove the residual culture medium components in the exosomes; the supernatant is removed, and the precipitate is resuspended in cold DPBS to obtain the engineered exosome drug delivery system overexpressing CD24.

[0007] As a further limitation of the second aspect of the present invention, the preparation of the plasmid overexpressing CD24 includes: cloning the Signal peptide fragment and the LAMP2B fragment from the GNSTM-3-Flag-10-Lamp2b-HA plasmid by PCR technology; cloning the Myc-CD24 fragment from the pCMV-Cd24-FLAG-Neo plasmid, wherein a GNSTM motif is added to the N-terminus of the Myc fragment in the Myc-CD24 fragment, and the Signal peptide fragment is fused to the N-terminus of the GNSTM motif; fusing the LAMP2B fragment to the C-terminus of the CD24 segment of the Myc-CD24 fragment by homologous recombination; ligating the fusion fragment of the LAMP2B fragment to the PLVX-IRES-Puro vector to construct a fusion expression plasmid of Signal Peptide-Myc-CD24-LAMP2B, that is, the plasmid overexpressing CD24.

[0008] In a third aspect, the present invention provides an anti-tumor drug encapsulated with a small molecule chemotherapeutic drug, using the engineered exosome drug delivery system overexpressing CD24 as described in the first aspect.

[0009] In a fourth aspect, the present invention provides a gene drug encapsulated with a gene editing tool, using the engineered exosome drug delivery system overexpressing CD24 as described in the first aspect.

[0010] Advantages of the present invention: By using the fusion gene encoding the transmembrane protein LAMP2B and CD24, an engineered exosome overexpressing CD24 on the exosome membrane was successfully constructed, which can effectively slow down its aggregation and protein adsorption phenomena, enhance the targeting of its drug delivery, and prolong its survival time in vivo, further improving its delivery performance.

[0011] Additional aspects and advantages of the present invention will be given in part in the following description, which will become apparent from the following description, or can be learned through the practice of the present invention. Description of the Drawings

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0013] Figure 1 Transmission electron microscope observation image of exosomes transfected with empty plasmid as described in the embodiments of the present invention.

[0014] Figure 2 Transmission electron microscope observation image of exosomes transfected with CD24L plasmid as described in the embodiments of the present invention.

[0015] Figure 3 Colony PCR result image of Signal Peptide-Myc-CD24-LAMP2B fusion protein as described in the embodiments of the present invention.

[0016] Figure 4 Colony PCR result image of CD24-EGFP-FLAG fusion protein as described in the embodiments of the present invention.

[0017] Figure 5 Electrophoresis images of Myc, FLAG, and β-antin expression in each group of cells as described in the embodiments of the present invention.

[0018] Figure 6 Particle size distribution image of exosomes transfected with empty plasmid as described in the embodiments of the present invention.

[0019] Figure 7 It is the particle size distribution diagram of the exosomes transfected with CD24L plasmid described in the embodiments of the present invention.

[0020] Figure 8 It is the electrophoresis diagram of the expression of Myc, FLAG and markers in the exosomes of each group described in the embodiments of the present invention.

[0021] Figure 9 It is the electrophoresis diagram of the expression of Myc, FLAG and CD63 in the membranes of the exosomes of each group described in the embodiments of the present invention.

[0022] Figure 10 It is the schematic diagram of nano flow cytometry analysis of CD24-EGFP exo described in the embodiments of the present invention.

[0023] Figure 11 It is the in vivo fluorescence imaging result diagram at different time points after intravenous injection of Control exo and CD24L exo (100 μg) into mice described in the embodiments of the present invention.

[0024] Figure 12 It is the whole body fluorescence quantitative analysis diagram of mice described in the embodiments of the present invention. Among them, n = 5. Compared with the control group, ns: p > 0.05, **: p < 0.01.

[0025] Figure 13 It is the HE staining result diagram of the main organs of mice after continuous intravenous injection of CD24L exo or DPBS for 12 days described in the embodiments of the present invention. Specific embodiments

[0026] The embodiments of the present invention provide an engineered exosome drug delivery system overexpressing CD24, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the scope of the present invention.

[0027] The present invention adopts genetic engineering technology and successfully constructs engineered exosomes (CD24L exo) overexpressing CD24 on the exosome membrane by using a fusion gene encoding transmembrane proteins LAMP2B and CD24. Among them, the CD24 molecule (Cluster of Differentiation 24, CD24) is a highly glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, which binds to the inhibitory receptor sialic acid-binding immunoglobulin-like lectin 10 (Siglec-10) on the membrane of tumor-associated macrophages, thereby avoiding macrophage phagocytosis. In addition, CD24 exists on the surface of exosomes. As an exosome marker, overexpressing CD24 on the exosome membrane to achieve its immune escape from the mononuclear macrophage system is an effective strategy to extend the survival time of exosomes in vivo. Lysosome-associated membrane protein 2B (LAMP2B) is an important component of the exosome membrane and can modify the molecules on the exosome surface.

[0028] In the present invention, the in vivo distribution and metabolic characteristics of CD24L exo (exosomes transfected with CD24L plasmid) as an in vivo drug delivery carrier were analyzed through in vivo distribution experiments, and its safety was evaluated. Compared with the control exosomes Control exo (exosomes transfected with empty plasmid), there were no obvious differences in their marker proteins, peak particle size, and shape; CD24L exo was distributed in the organ tissues of mice, and the in vivo fluorescence intensity at 48 h was significantly higher than that of Control exo (p < 0.01); continuous administration of 100 μg CD24L exo for 12 days did not cause damage to the important organs of mice. Therefore, the CD24L exo constructed in the present invention has stronger immune escape ability, longer in vivo survival time, and good safety compared with Control exo.

[0029] Specifically, in the specific embodiments of the present invention, two plasmids were prepared, namely the fusion expression plasmid of SignalPeptide-Myc-CD24-LAMP2B (CD24L plasmid) and the fusion expression plasmid of CD24-EGFP -FLAG (CD24-EGFP plasmid), and the expression performance and characteristics of the two plasmids were verified through comparative experiments.

[0030] In a specific embodiment, the methods for preparing the above two plasmids are as follows: The Signal Peptide fragment and the LAMP2B fragment were respectively cloned from the GNSTM-3-Flag-10-Lamp2b-HA plasmid by PCR; the Myc-CD24 fragment was cloned from the pCMV-Cd24-FLAG-Neo plasmid, wherein the GNSTM motif was added to the N-terminus of the Myc fragment in the Myc-CD24 fragment, and the Signal peptide fragment was fused to the N-terminus of the GNSTM motif; the LAMP2B fragment was fused to the C-terminus of the CD24 fragment in the Myc-CD24 fragment by homologous recombination; finally, the above fragments were ligated into the PLVX-IRES-Puro vector to construct a plasmid for fusion expression of Signal Peptide-Myc-CD24-LAMP2B (CD24L plasmid, that is, a plasmid overexpressing CD24). The CD24 fragment was cloned from the pCMV-Cd24-FLAG-Neo plasmid, and the EGFP-FLAG fragment was cloned from the PLVX-EGFP-Puro vector. The CD24 fragment was fused to the N-terminus of EGFP-FLAG by homologous recombination to construct a plasmid for fusion expression of CD24-EGFP -FLAG (CD24-EGFP plasmid).

[0031] In this example, the PCR reaction system constructed in the above PCR process was: 2×Phanta Mix Buffer, 25 μL; Phanta Mix Super Fidelity, 1 μL; dNTP Mix (10 mM each), 1 μL; template plasmid, 1 μL (10 ng); upstream primer, 2 μL; downstream primer, 2 μL; double-distilled water, 18 μL. The PCR reaction procedure was: after the above system was configured and centrifuged briefly, amplification was carried out in a PCR instrument. The PCR instrument used in this example was of the type from Bio-Rad. The PCR amplification conditions were set as follows: (1) Pre-denaturation: 95°C for 3 min; (2) Denaturation: 95°C for 20 s; (3) Annealing: 55°C for 20 s; (4) Extension: 72°C for 90 s; (5) Final extension: 72°C for 5 min. Among them, one cycle was completed by performing the denaturation, annealing, and extension steps once, and the number of cycles was set to 30. The remaining steps were performed once.

[0032] In this example, the sequences of the prepared CD24L plasmid and CD24-EGFP plasmid are shown in SEQ ID No. 1 and SEQ ID No. 2 respectively. In the above PCR process, the upstream primer sequence for cloning the Signal Peptide fragment is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No. 4; the upstream primer sequence for cloning the Myc fragment is shown in SEQ ID No. 5, and the downstream primer sequence is shown in SEQ ID No. 6; the upstream primer sequence for cloning the LAMP2B fragment is shown in SEQ ID No. 7, and the downstream primer sequence is shown in SEQ ID No. 8; the upstream primer sequence for cloning the CD24 fragment is shown in SEQ ID No. 9, and the downstream primer sequence is shown in SEQ ID No. 10; the upstream primer sequence for cloning the EGFP-FLAG fragment is shown in SEQ ID No. 11, and the downstream primer sequence is shown in SEQ ID No. 12.

[0033] In this example, three types of exosomes constructed with CD24L plasmid, CD24-EGFP plasmid and empty plasmid were extracted respectively, and the performance of CD24L plasmid was verified through comparative experiments.

[0034] Specifically, the extraction and preparation methods of the above three types of exosomes are as follows: Mix 1 μg of CD24 target plasmid (CD24L plasmid), CD24-EGFP plasmid and empty plasmid (Control) with 160 μL of Opti-MEM, 0.75 μg of psPAX2, 0.5 μg of pMD2.G and 10 μL of PEI transfection reagent respectively. After mixing evenly, add 167 μL per well to a 6-well plate. Harvest the cell supernatants after 48 h and 72 h respectively. After infecting HEK293T cells with the corresponding supernatants for 72 h, transfer them to a 10 cm dish for normal culture. When the cell confluence reaches about 90%, culture for another 24 h. Collect the culture supernatants of HEK293T cells, Control HEK293T cells transfected with empty plasmid, CD24LHEK293T cells transfected with CD24L plasmid and CD24-EGFP HEK293T cells transfected with CD24-EGFP plasmid. Centrifuge at 4000×g for 30 min at 4 °C to remove cells and cell debris, then filter with a 0.22 μm filter membrane, and centrifuge the supernatant at 120000×g for 120 min to granulate the exosomes, discard the supernatant; resuspend the exosome particles in 1 mL of DPBS and further ultracentrifuge at 120000×g for 120 min to remove the residual culture medium components in the exosomes; remove the supernatant, resuspend the exosome precipitate with 1 mL of cold DPBS and store at -80 °C.

[0035] In this embodiment, the process of exosome characterization, marker protein identification and labeling is as follows: Transmission Electron Microscope (TEM) is used to characterize the state of exosome particles: Take 10 μL of the exosome resuspension and drop it on the copper grid for loading samples. After precipitation at room temperature for 2 min to 3 min, use filter paper to absorb the floating liquid at the edge of the copper grid, then negatively stain with an equal volume of 3% phosphotungstic acid solution for 4 min, and then use filter paper to absorb the negative stain solution. After drying at room temperature for 3 min, perform on-machine image acquisition, and adjust the electron microscope acceleration voltage to 100 kv. The images of two types of exosome particles, Control exo and CD24L exo, obtained by TEM characterization are respectively as Figure 1 , Figure 2 shown.

[0036] The diameter of exosome particles is measured by Nanoparticle Tracking Analysis (NTA) technology: Dilute the exosome sample to 1×10 9 per mL and then inject it into the sample cell; Set the instrument parameters and start the detection to obtain the particle size distribution and concentration.

[0037] Identification of exosome marker proteins: Add RIPA lysis buffer to the exosomes of the obtained Control HEK293T cells, CD24L HEK293T cells and CD24-EGFP HEK293T cells respectively, and lyse them on ice for 15 min. Then, measure the protein concentration by the BCA method, and perform Western blot detection on the exosome marker proteins CD9 molecule (Cluster of Differentiation 9, CD9), CD63 molecule (Cluster of Differentiation 63, CD63), and Tumor Susceptibility Gene 101 (TSG101). Among them, Control HEK293T cells, CD24L HEK293T cells and CD24-EGFP HEK293T cells are all cultured in DMEM medium containing 10% fetal bovine serum. The fetal bovine serum is centrifuged at 120000×g for 16 h to remove exosomes and then used for cell culture.

[0038] Exosome labeling: The purified exosomes were incubated at 37 °C for 10 min in the presence of 2.5 μmol / L Dil, and then ultracentrifuged at 120,000×g for 90 min to remove the residual Dil dye. After centrifugation at 120,000×g, the exosomes were washed once with DPBS, and the supernatant was removed. The labeled exosomes were resuspended with DPBS or DMEM before use.

[0039] Extraction of exosome membrane proteins: 1 mL of Membrane Protein Extraction Reagent A supplemented with protease inhibitor was added to 5 mg of exosomes, gently suspended, and placed on ice for 10 - 15 min. Subsequently, the exosomes were thoroughly disrupted, and impurities were removed by centrifugation at 700×g for 10 min at 4 °C. The supernatant was carefully collected. Then, the exosome membrane fragments were precipitated by centrifugation at 14,000×g for 30 min at 4 °C. The supernatant was removed, and the precipitate was resuspended with Membrane Protein Extraction Reagent B and vortexed vigorously and incubated on ice once or twice. The supernatant was collected by centrifugation again to obtain the exosome membrane protein solution, which was stored at -80 °C for later use.

[0040] Experiment on in vivo distribution, metabolism and safety analysis of exosomes: Detection of in vivo distribution of exosomes by small animal in vivo imager: Ten nude mice were randomly divided into a Control group and a CD24L group according to a random number table, with 5 mice in each group. Approximately 100 μg of Control exo and CD24Lexo labeled with Dil were respectively injected into the nude mice via the tail vein. The mice were imaged using a small animal in vivo imager at 3 h, 24 h, and 48 h to observe the distribution of exosomes in the nude mice.

[0041] Ten female BALB / c nude mice were randomly divided into 2 groups according to a random number table. One group was injected with CD24Lexo intravenously at a dose of 100 μg per day for 12 consecutive days, and the other group was used as a control with DPBS. At 24 h after the last administration, major organs such as the heart, liver, spleen, lung, and kidney were taken for immunohistochemical analysis. After baking the paraffin sections at 75 °C for 1 h, they were immediately placed in xylene, successively in xylene I and xylene II for at least 15 - 20 min, and then successively in absolute ethanol, 95%, 85%, and 75% ethanol for about 5 min each time, followed by washing with distilled water for dewaxing to water treatment. After washing with water, the sections were successively stained with hematoxylin for nuclei, differentiated with 1% hydrochloric acid ethanol, blued with 0.5% ammonia water, and stained with eosin staining solution for cytoplasm. Each staining interval was washed with tap water, and then dehydration and clearing were completed in the reverse order of the dewaxing to water steps. After slightly drying, the sections were sealed with neutral gum. The stained sections were observed, images were collected, and analysis was performed under a microscope.

[0042] The experimental results were graphed and analyzed using GraphPad Prism 8.0 software, and the experimental results were expressed as mean + SEM. The t-test was used between two data sets. Statistically significant differences were expressed as *p < 0.05 and **p < 0.01.

[0043] In summary, two CD24 expression plasmids, namely CD24L plasmid and CD24-EGFP plasmid, were successfully constructed in the embodiments of the present invention. As Figure 3 、 Figure 4 shown, the CD24L plasmid and the CD24-EGFP plasmid were transfected into HEK293T cells to prepare a stable cell line. After collecting the corresponding cells, total protein was extracted and verified by Western blot using Myc tag and FLAG tag antibodies, respectively, and compared with Control HEK293T. The results showed that Myc and FLAG were highly expressed in the corresponding cells, indicating that the CD24L and CD24-EGFP plasmids had been transferred into HEK293T cells and successfully expressed, as Figure 5 shown.

[0044] In this example, the identification of engineered exosomes overexpressing CD24 was as follows: The NTA detection results showed that Control exo and CD24L exo had a uniform size, with a peak at around 115 nm, indicating that there was no significant difference in their sizes; TEM observation showed no obvious difference in the morphology of Control exo and CD24L exo, as Figure 6 、 Figure 7 shown; The exosomal marker proteins CD9, CD63, and TSG101 of CD24L exo and CD24-EGFP exo were highly expressed, as Figure 8 shown. The above identification results indicated that two kinds of engineered exosomes were successfully prepared in the embodiments of the present invention.

[0045] Total protein of CD24L exo and CD24-EGFP exo was extracted and verified by Western blot using Myc tag and FLAG tag antibodies, respectively. Compared with the empty exosomes (Control exo) obtained by transfection with the empty plasmid, Myc tag was highly expressed while FLAG tag was not significantly expressed, as Figure 8 shown.

[0046] It is illustrated that after transfection of HEK293T cells with CD24-EGFP plasmid, only CD24 was overexpressed in the cells, while after transfection of cells with CD24L plasmid, CD24 was overexpressed both in the cells and exosomes. To further verify whether CD24 is expressed on the exosome membrane, the membrane proteins of CD24L exo and CD24-EGFP exo were extracted, and it was found that the Myc tag was highly expressed on the exosome membrane, while the FLAG tag was hardly expressed, as Figure 9 shown; the expression of CD24-EGFP in CD24-EGFP exo was identified by nano flow cytometry, and the expression rate was 6.6%, as Figure 10 shown.

[0047] The above results suggest that compared with the CD24-EGFP plasmid, the CD24L plasmid can significantly increase and achieve the expression of CD24 on the exosome membrane.

[0048] The in vivo distribution of CD24L exo was further evaluated using an in vivo imaging system, as Figure 11 shown. After 3 h of receiving Dil-labeled CD24L exo and Control exo injected via the tail vein, the distribution of the two exosomes throughout the body of the mice mainly concentrated in the lungs, liver, kidneys and other parts, and there was no significant difference in the distribution; after 24 h to 48 h of injection, the fluorescence intensity of CD24L exo in the mice was significantly higher than that of Control exo (p < 0.01), as Figure 12 shown. The above results indicate that compared with Control exo, CD24L exo has a longer survival time in vivo.

[0049] CD24L exo was continuously injected into healthy BALB / c nude mice via the tail vein, and no death occurred during the whole process. In the mice receiving CD24L exo injection, the myocardial cells were arranged orderly, the hepatic lobule structure was intact, the lymphoid follicles of the spleen were clearly visible, the alveolar structure was intact, and there were no obvious abnormalities in the structure of renal tubules and glomeruli, showing no significant difference compared with the DPBS group, as Figure 13 shown. It shows that CD24L exo has no damage to the main organ tissues and has good safety.

[0050] In summary, the body's natural clearance mechanism significantly affects the metabolic kinetics of exosomes, which is an important challenge faced by exosome drug delivery carriers in clinical translation. The half-life of exosomes derived from mouse plasma is only about 7 minutes, while exosomes derived from tumors are cleared from the circulatory system even faster, with a half-life of 2 minutes. This is mainly attributed to the extensive clearance by monocytes / macrophages or the reticuloendothelial system (RES) in the blood. Tumor cells play an important role in the recognition of the mononuclear phagocytic system through immune checkpoint axes such as CD24-Siglec-10, CD47 molecule-Signal regulatory protein alpha (Cluster of Differentiation 47-Signalregulatory protein alpha, CD47-SIRPα), Programmed Death-Ligand 1-Programmed Cell Death Protein 1 (Programmed Death-Ligand 1-Programmed Cell Death Protein 1, PDL1-PD1), β-2-Microglobulin-Leukocyte Immunoglobulin-like Receptor Subfamily B Member 1 (β-2-Microglobulin-Leukocyte Immunoglobulin-like Receptor Subfamily B Member 1, β2M-LILRB1), etc.

[0051] In the embodiments of the present invention, the above mechanism is applied to the engineering modification of exosomes to construct a CD24-Siglec-10-mediated exosome escape system. By overexpressing CD24 on the surface of exosome membranes, the immune escape of engineered exosomes CD24Lexo from macrophages is effectively achieved. Fluorescence imaging results show that the retention time of CD24L exo in BALB / c nude mice is significantly prolonged compared with the control group (p < 0.01), and a relatively high signal intensity is still maintained at 48 h, laying an important foundation for improving the in vivo delivery efficiency of the drug delivery system.

[0052] In the embodiments of the present invention, the exosomes obtained by transfecting cells with the CD24L plasmid prepared above are surface-modified to endow them with specific functions and can become drug delivery carriers.

[0053] In a specific embodiment, the engineered exosomes overexpressing CD24 are obtained by transfecting cells with the above-prepared CD24L plasmid, which can be applied to the preparation of anti-tumor drugs. By encapsulating small molecule chemotherapeutic drugs inside them, they can be used as an anti-tumor drug. For example, when applied to the preparation of drugs for treating triple-negative breast cancer, taxanes, anthracyclines or platinum drugs can be encapsulated inside as chemotherapeutic drugs, and encapsulating PD-1 / PD-L1 inhibitors can be used as drugs for immunotherapy, and encapsulating PARP inhibitors and AKT inhibitors can be used as drugs for targeted therapy.

[0054] In another specific embodiment, the engineered exosomes overexpressing CD24 are obtained by transfecting cells with the above-prepared CD24L plasmid. By encapsulating gene editing tools inside them, they can be used as a gene drug. For example, when applied to the preparation of drugs for treating hemophilia, coagulation factor genes can be encapsulated inside as gene drugs for treating hemophilia.

[0055] In another specific embodiment, the engineered exosomes overexpressing CD24 obtained by transfecting cells with the above-prepared CD24L plasmid can also be applied to the preparation of drugs for inhibiting cancer cell migration. For example, drugs for inhibiting the migration of human lung adenocarcinoma epithelial cells.

[0056] In the present invention, the overexpressing plasmid CD24-EGFP fails to effectively enrich CD24 on the exosome membrane structure, which is presumably related to insufficient post-translational modification regulation of the GPI transamidase complex. Therefore, the present invention proposes a targeting and anchoring strategy based on LAMP2B. LAMP2B is a typical type I transmembrane protein structure that is widely present on the exosome membrane and contains a long chain. Its N-terminus is highly glycosylated and located on the lumen side of the membrane, while the C-terminus is exposed inside the lumen. The protein fused to the N-terminus of LAMP2B exhibits high surface expression characteristics and does not affect the expression and function of LAMP2B. Therefore, the CD24 sequence is fused to the N-terminus of LAMP2B. At the same time, to prevent the expressed peptide from being degraded by acid-dependent hydrolytic proteins, a glycosylation sequence GNSTM is introduced at the N-terminus of the fusion sequence. The steric hindrance effect is used to provide protection for the peptide, thereby enhancing the stability of the LAMP2B-CD24 fusion protein. At the same time, the Signal peptide fragment of LAMP2B can localize the fusion protein to lysosomes to guide the successful localization of the fusion protein to exosomes. The results show that compared with the traditional overexpressing plasmid, the expression efficiency of CD24 on the exosome membrane surface has been significantly improved for the fusion plasmid (i.e., CD24 plasmid) constructed in the present invention. TEM and NTA detection confirm that the engineering transformation does not change the natural biological characteristics such as the morphology, particle size distribution and marker protein expression of exosomes. In terms of biosafety, histopathological evaluation preliminarily proves that continuous injection of CD24L exo for 12 days does not cause pathological damage to the important organs of mice, indicating good short-term safety.

[0057] Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, they are not limitations on the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solutions disclosed in the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts should be covered within the protection scope of the present invention.

Claims

1. An engineered exosome drug delivery system overexpressing CD24, characterized in that, The membrane of the engineered exosomes overexpresses CD24, and the gene sequence of the plasmid overexpressing CD24 is shown in SEQ ID No.

1.

2. The engineered exosome drug delivery system overexpressing CD24 according to claim 1, wherein The N-terminus of the type I transmembrane protein structure LAMP2B on the membrane of the engineered exosomes is fused with the CD24 sequence, a glycosylation sequence GNSTM is introduced at the N-terminus of the fusion sequence, and the N-terminus of GNSTM is fused with the exosome-binding peptide Signal peptide fragment.

3. The engineered exosome drug delivery system overexpressing CD24 according to claim 2, wherein The plasmid overexpressing CD24 includes adding a GNSTM motif to the N-terminus of the Myc fragment in the Myc-CD24 fragment, the N-terminus of the GNSTM motif is fused with the Signal peptide fragment, and the C-terminus of the CD24 fragment in the Myc-CD24 fragment is fused with the LAMP2B fragment; among them, the Signal peptide fragment and the LAMP2B fragment are cloned from the GNSTM-3-Flag-10-Lamp2b-HA plasmid by PCR technology, and the Myc-CD24 fragment is cloned from the pCMV-Cd24-FLAG-Neo plasmid.

4. A method for preparing an engineered exosome drug delivery system overexpressing CD24 according to any one of claims 1 to 3, characterized in that, The plasmid overexpressing CD24 is mixed evenly with Opti-MEM, psPAX2, pMD2.G and PEI transfection reagent and then added to the well plate, the cell supernatant is obtained, HEK293T cells are infected, transferred to a dish for normal culture, the culture supernatant of the cells is collected, centrifuged to remove the cells and cell debris, the filtered supernatant is centrifuged to granulate the exosomes, and the supernatant is discarded; the exosome particles are resuspended in DPBS, ultracentrifuged to remove the residual culture medium components in the exosomes; the supernatant is removed, precipitated, and resuspended in cold DPBS to obtain the engineered exosome drug delivery system overexpressing CD24.

5. The preparation method of the engineered exosome drug delivery system overexpressing CD24 according to claim 4, characterized in that, The preparation of the plasmid overexpressing CD24 includes: cloning the Signal peptide fragment and the LAMP2B fragment from the GNSTM-3-Flag-10-Lamp2b-HA plasmid by PCR technology; cloning the Myc-CD24 fragment from the pCMV-Cd24-FLAG-Neo plasmid, wherein a glycosylation sequence GNSTM is added to the N-terminus of the Myc fragment in the Myc-CD24 fragment, and the N-terminus of GNSTM is fused with the Signal peptide fragment; fusing the LAMP2B fragment to the C-terminus of the CD24 segment of the Myc-CD24 fragment by homologous recombination; connecting the fusion fragment of the LAMP2B fragment to the PLVX-IRES-Puro vector to construct a fusion expression Signal Peptide-Myc-CD24-LAMP2B plasmid, that is, the plasmid overexpressing CD24.

6. An anti-tumor drug, characterized in that, Using the engineered exosome drug delivery system overexpressing CD24 according to any one of claims 1 to 3, a small molecule chemotherapeutic drug is encapsulated.

7. A gene drug, characterized in that, Using the engineered exosome drug delivery system overexpressing CD24 according to any one of claims 1 to 3, a gene editing tool is encapsulated.

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