Immune escape exosome and preparation method thereof

Abstract

The application relates to the field of biological medicine, and provides an immune escape exosome and a preparation method thereof, wherein a chimera molecule CD47-CD63 overexpression human lung bronchial epithelial cell HBE and a mouse embryonic fibroblast cell NIH / 3T3 cell strain are constructed through genetic engineering technology, and an exosome rich in the chimera molecule CD47-CD63 protein is separated and purified, the immune escape exosome prepared by the application can be used as a nano drug or loaded with various drugs, can significantly escape phagocytosis and clearance of macrophages and an in-vivo immune system, and can further effectively overcome the key bottleneck problems of short blood circulation half-life, low delivery efficiency, large exosome aggregation in tissues such as liver and spleen, relatively small arrival at a lesion, and non-obvious treatment effect of the current exosome nano drug or exosome drug delivery carrier.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an immune escape exosome and its preparation method. Background Technology

[0002] A drug delivery system (DDS) refers to a technological system that allows for comprehensive control over the distribution of drugs within a biological organism in terms of time, space, and dosage. During the development of nanotechnology, a wide variety of multifunctional nanomaterials have been discovered and fabricated, finding extensive applications in the medical field. The most commonly used nanoparticle drug delivery systems are liposomes and polymer nanoparticles. Liposome nanoparticles are synthetic vesicles with phospholipid membranes that can self-assemble into various sizes and shapes in an aqueous environment; polymer nanoparticles are a drug delivery system that facilitates the encapsulation, sealing, or attachment of drug molecules. Both delivery systems are used to deliver different types of drug molecules, including anticancer drugs, antifungal drugs, and analgesics. However, the toxicity of liposomes and the biocompatibility of polymer nanoparticles are significant drawbacks that are difficult to overcome in drug delivery carriers.

[0003] While nanoparticles offer significant advantages, they also present challenges. Nanoparticles obtained through traditional chemical methods suffer from drawbacks such as high fusion rates, strong immunogenicity, low stability, and cytotoxicity, posing new challenges to drug delivery research. Exosomes, cellular secretion products, offer a solution to these shortcomings to some extent as direct nanomedicines or drug delivery carriers. They combine the advantages of nanoparticles with their inherent limitations, making them a promising biopharmaceutical or drug delivery vehicle. Although homologous exosomes can significantly reduce the toxicity of physicochemical materials exposed to the body, they cannot prevent rapid immune clearance by the mononuclear cell phagocytic system. Consequently, they have a short blood circulation half-life, low delivery efficiency, and a high accumulation of exosomes in the liver and spleen, resulting in relatively less drug reaching the lesion and limited therapeutic efficacy. These are key issues hindering the use of exosomes as nanomedicines or drug delivery carriers. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the related art. To this end, the present invention provides a method for preparing immune escape exosomes, comprising the following steps:

[0005] S1: Based on the CD47 and CD63 gene CDS sequences, using HBE cell cDNA as a template, the full-length cDNA of CD47 and CD63 genes was cloned by PCR to obtain the full-length cDNA of CD47 and CD63.

[0006] S2: Based on the full-length cDNA of CD47 and CD63, the sequence of the CD47 immune escape domain and the sequence of the CD63 exosome enrichment domain were determined.

[0007] S3: The CD47 immune escape domain sequence and the CD63 exosome enrichment domain sequence were cloned a second time to obtain the chimeric molecule CD47-CD63.

[0008] S4: The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 is packaged with viruses to obtain a lentivirus overexpressing the chimeric molecule CD47-CD63.

[0009] S5: Human bronchial epithelial cells (HBE) and mouse embryonic fibroblasts (NIH / 3T3) were infected with lentiviruses that overexpressed the chimeric molecule CD47-CD63 to obtain cell lines that stably overexpressed CD47-CD63 protein.

[0010] S6: The CD47-CD63 protein stably overexpressing cell line is subjected to serum-free cell culture to obtain cell culture supernatant. The cell culture supernatant is then subjected to differential centrifugation and ultracentrifugation to obtain immune escape exosomes.

[0011] According to the method for preparing immune escape exosomes provided by the present invention, the steps of step S2 are as follows:

[0012] S21: Using the full-length cDNA of CD47 as a template, PCR cloning and amplification were performed using primers specific to the CD47 immune escape domain sequence containing the CD63 adapter sequence to obtain linear DNA encoding the CD47 immune escape domain.

[0013] S22: Using the full-length cDNA of CD63 as a template, PCR cloning and amplification were performed using primers specific to the CD63 exosome enrichment domain sequence with CD47 adapter sequence to obtain linear DNA encoding the CD63 exosome enrichment domain.

[0014] According to the method for preparing immune escape exosomes provided by the present invention, the steps of step S3 are as follows:

[0015] S31: Using the CD47 immune escape domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD47 pre-primer to obtain the first linear DNA.

[0016] S32: Using the CD63 exosome enrichment domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD63 back primer to obtain the second linear DNA.

[0017] S33: The first linear DNA and the second linear DNA are spliced ​​together to obtain the linear DNA of the chimeric molecule CD47-CD63.

[0018] According to the method for preparing immune escape exosomes provided by the present invention, step S4 is as follows:

[0019] The linear DNA of the chimeric molecule CD47-CD63 was digested and ligated with enzymes, and then inserted into the lentiviral expression vector pLV-EF1α-MCS-IRES Puro to obtain a lentiviral expression plasmid vector of the chimeric molecule CD47-CD63. The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 and the packaging plasmid were co-transfected into 293T cells to obtain lentivirus overexpressing the chimeric molecule CD47-CD63.

[0020] According to the method for preparing immune escape exosomes provided by the present invention, step S5 is as follows:

[0021] Lentiviral cells overexpressing the chimeric molecule CD47-CD63 were mixed with antibiotic-free medium at a 1:1 ratio to obtain a mixed medium. Polybrane was added to the mixed medium to culture HBE and NIH / 3T3 cells. After 12 hours, HBE and NIH / 3T3 cells were placed in basal medium and cultured for another 48 hours. Then, puro-resistant medium was added to the basal medium to screen and culture HBE and NIH / 3T3 cells to obtain a stable CD47-CD63 protein overexpressing cell line.

[0022] According to the present invention, a method for preparing immune escape exosomes is provided, wherein the basal culture medium is a culture medium supplemented with 1% antibiotics and 10% fetal bovine serum.

[0023] According to the method for preparing immune escape exosomes provided by the present invention, step S6 is as follows:

[0024] S61: The HBE and NIH / 3T3 cell lines that stably overexpress the CD47-CD63 protein were cultured in basal medium until the cell density reached approximately 80%.

[0025] S62: Wash the HBE and NIH / 3T3 cells, which were at a density of approximately 80%, with PBS, then add serum-free medium to the washed cells and culture for 36 hours.

[0026] S63: Collect the cell culture supernatant after 36 hours, centrifuge the cell culture supernatant at differential speed, then ultrafilter it with a nanoporous membrane, and finally separate and purify the exosomes in the cell culture supernatant by ultracentrifugation to obtain immune escape exosomes.

[0027] An immune escape exosome prepared according to a method for preparing immune escape exosomes provided by the present invention contains chimeric molecules CD47-CD63.

[0028] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0029] This invention provides an immune-escape exosome and its preparation method. Human bronchial epithelial cells (HBE) and mouse embryonic fibroblasts (NIH / 3T3) overexpressing the chimeric molecule CD47-CD63 are constructed using genetic engineering technology. Exosomes enriched with the chimeric molecule CD47-CD63 protein are isolated, purified, and enriched. The immune-escape exosomes prepared by this invention can be used as nanomedicines or loaded with multiple drugs. In vitro experiments show that exosomes enriched with the chimeric molecule CD47-CD63 protein can significantly evade phagocytosis and clearance by macrophages. In vivo experiments in mice show that exosomes enriched with the chimeric molecule CD47-CD63 protein can significantly evade clearance by the in vivo immune system. This invention effectively overcomes the key bottleneck problems of current exosomal nanomedicines or exosomal drug delivery carriers, such as short blood circulation half-life, low delivery efficiency, and large accumulation of exosomes in tissues such as the liver and spleen, resulting in relatively few reaching the lesion and insignificant therapeutic effects.

[0030] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 Electrophoresis diagram of the full-length cDNA sequence of the CD63 gene.

[0033] Figure 2 Electrophoresis diagram of the full-length cDNA sequence of the CD47 gene.

[0034] Figure 3 Electrophoresis diagram of the cDNA nucleic acid sequence of the chimeric molecule CD47-CD63.

[0035] Figure 4 This is a Western blot diagram showing the stable overexpression of chimeric molecules CD47-CD63 and CD47 in HBE cells.

[0036] Figure 5 Western blot diagram showing the stable overexpression of chimeric molecules CD47-CD63 and CD47 in NIH / 3T3 cells.

[0037] Figure 6 Electron micrographs comparing HBE cell-derived exosomes that are wild-type, overexpress CD47, and contain the chimeric molecule CD47-CD63.

[0038] Figure 7 Electron micrographs comparing exosomes derived from NIH / 3T3 cells that are wild-type, overexpress CD47, and contain the chimeric molecule CD47-CD63.

[0039] Figure 8 This is a Western blot diagram showing the induction of M1 macrophages from Raw 264.7 cells using LPS.

[0040] Figure 9 This is an immunofluorescence experiment showing how LPS can induce Raw 264.7 cells to become M1 macrophages.

[0041] Figure 10 Comparative immunofluorescence assay of M1 macrophages phagocytosing wild-type HBE cells, HBE cells overexpressing CD47, and HBE cells with the chimeric molecule CD47-CD63.

[0042] Figure 11 The average fluorescence intensity of M1 macrophages phagocytosing wild-type HBE cells, cells overexpressing CD47, and the chimeric molecule CD47-CD63 is quantitatively represented.

[0043] Figure 12 Comparative immunofluorescence assay of M1 macrophages phagocytosing wild-type NIH / 3T3 cells, cells overexpressing CD47, and the chimeric molecule CD47-CD63.

[0044] Figure 13 This is a quantitative graph of the mean fluorescence intensity of M1 macrophages phagocytosing wild-type, CD47-overexpressing, and chimeric molecule CD47-CD63 NIH / 3T3 cells.

[0045] Figure 14 Comparative Western blot diagram of exosomal proteins derived from HBE cells in wild-type cells, cells overexpressing CD47, and cells containing the chimeric molecule CD47-CD63.

[0046] Figure 15Comparative Western blot results of exosomal proteins derived from NIH / 3T3 cells with wild-type cells, cells overexpressing CD47, and cells containing the chimeric molecule CD47-CD63.

[0047] Figure 16 Immunofluorescence assay comparing M1 macrophages phagocytizing wild-type HBE cell-derived exosomes that overexpress CD47 and the chimeric molecule CD47-CD63.

[0048] Figure 17 This is a quantitative graph of the mean fluorescence intensity of HBE cell-derived exosomes phagocytosed by M1 macrophages, which are wild-type, overexpressing CD47, and containing the chimeric molecule CD47-CD63.

[0049] Figure 18 Immunofluorescence assay comparing M1 macrophages phagocytizing wild-type, CD47-overexpressing, and chimeric molecule CD47-CD63 exosomes derived from NIH / 3T3 cells.

[0050] Figure 19 This is a quantitative graph of the mean fluorescence intensity of NIH / 3T3 cell-derived exosomes that are phagocytosed by M1 macrophages on wild-type, CD47-overexpressing, and chimeric molecule CD47-CD63.

[0051] Figure 20 Comparative image of single-particle tracking experiments on the surface of M1 macrophages using exosomes derived from HBE cells that are wild-type, overexpressing CD47, and the chimeric molecule CD47-CD63.

[0052] Figure 21 A comparative diagram showing the tracking trajectories of HBE cell-derived exosomes, including wild-type, CD47-overexpressing, and chimeric molecule CD47-CD63, on the surface of M1 macrophages.

[0053] Figure 22 Comparative image of single-particle tracking assay on the surface of M1 macrophages for exosomes derived from NIH / 3T3 cells that are wild-type, overexpressing CD47, and the chimeric molecule CD47-CD63.

[0054] Figure 23 A comparative image showing the tracking trajectories of wild-type, CD47-overexpressing, and CD47-CD63 chimeric molecule-derived exosomes from NIH / 3T3 cells on the surface of M1 macrophages.

[0055] Figure 24 Comparative diagram of NIH / 3T3 cell-derived exosomes phagocytosed by hematopoiesis of wild-type, CD47-overexpressing, and chimeric molecules CD47-CD63.

[0056] Figure 25This is a quantitative graph of the mean fluorescence intensity of NIH / 3T3 cell-derived exosomes that have been phagocytosed by hematopoiesis of wild-type, CD47-overexpressing, and chimeric molecules CD47-CD63.

[0057] Figure 26 This is a comparative experiment showing the residue of NIH / 3T3 cell-derived exosomes (wild-type, CD47-overexpressing, and the chimeric molecule CD47-CD63) after circulating in mice for 10 minutes.

[0058] Figure 27 This is a statistical chart showing the number of NIH / 3T3 cell-derived exosomes that are wild-type, overexpressing CD47, and the chimeric molecule CD47-CD63, remaining in mice after 10 minutes of circulation.

[0059] Figure 28 This is a comparative diagram showing the multi-organ distribution of NIH / 3T3 cell-derived exosomes (wild-type, CD47-overexpressing, and the chimeric molecule CD47-CD63) after circulating in mice for 60 minutes.

[0060] Figure 29 This image shows a quantitative comparison of fluorescence intensity distribution in multiple organs of NIH / 3T3 cell-derived exosomes (wild-type, CD47-overexpressing, and CD47-CD63 chimeric molecules) after 60 minutes of in vivo circulation in mice. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention but should not be used to limit the scope of this invention.

[0062] Furthermore, the terms “first,” “second,” and “third” are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0063] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0064] The following is in conjunction with the appendix Figures 1-29 The present invention describes an immune escape exosome and its preparation method:

[0065] This invention provides a method for preparing immune escape exosomes, comprising the following steps:

[0066] S1: Based on the CD47 and CD63 gene CDS sequences, using HBE cell cDNA as a template, the full-length cDNA of CD47 and CD63 genes was cloned by PCR to obtain the full-length cDNA of CD47 and CD63.

[0067] CD63 is a tetraspanin molecule belonging to the four-transmembrane protein family. Its N-terminus and C-terminus are both located inside the cell. As a molecular marker of exosomes, CD63 is widely used as a target for modifying exosome function by modifying specific functional peptides or protein molecules on the surface of exosomes. The phagocytic function of mononuclear phagocytes in the immune system is dynamically regulated by phagocytic promotion and inhibition, i.e., the "eat me" and "don't eat me" signaling system. The membrane protein CD47 is the representative of the "don't eat me" signal of cells. It has the function of inhibiting the phagocytosis of healthy cells and various cancer cells by the innate immune system. The SIRPα-CD47 axis constitutes the "don't eat me" signaling system of the innate immune response, which regulates the body's immune clearance system.

[0068] S2: Based on the full-length cDNA of CD47 and CD63, the sequence of the CD47 immune escape domain and the sequence of the CD63 exosome enrichment domain were determined.

[0069] like Figures 1 to 2 As shown, Figure 1 This is an electrophoresis image of the full-length cDNA sequence of the CD63 gene. The full-length CD63 cDNA consists of 717 base pairs, and the electrophoretic band is located between 750 bp and 1 kb in the DNA ladder. Figure 1 The area selected by the black box in the middle; Figure 2This is an electrophoresis image of the full-length cDNA sequence of the CD47 gene. The full-length CD47 cDNA consists of 969 base pairs, and the electrophoretic band is located approximately 1 kb from the DNA ladder. Figure 2 The area highlighted in the white box indicates that the N-terminal extracellular domain of the CD47 molecule is its key immunosuppressive functional domain.

[0070] The full-length cDNA sequence of CD47 with the Flag tag is as follows:

[0071] 。

[0072] The nucleotide sequence of the full-length cDNA of CD63 with Flag tag is:

[0073] AtggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccagggggctacccctggctctctgttgccagtggtcatcatcgcagtgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttccggcagcagatggagaattacccgaaaaacaaccacactgcttcgatcctggacaggatgcaggcagattttaagtgctgtggggctgctaactacacagattgggagaaaatcccttccatgtcgaagaaccgagtccccgactcctgctgcattaatgttactgtgggctgtgggattaatttcaacgagaaggcgatccataaggagggctgtgtggagaagattgggggctggctgaggaaaaatgtgctggtggtagctgcagcagcccttggaattgcttttgtcgaggttttgggaattgtctttgcctgctgcctcgtgaagagtatcagaagtggctacgaggtgatgATTACAAGGATGACGACGATAAG。

[0074] The amino acid sequence of the CD47 protein with Flag tag is:

[0075] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIF AILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLLVTSTGILILLHYYVFSTAIIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVEDYKDDDDK.

[0076] The amino acid sequence of the CD63 protein with the flag tag is as follows:

[0077] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQM ENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMDYKDDDDK.

[0078] Furthermore, the second transmembrane region and subsequent segments of the CD63 protein are key functional domain sequences for its enrichment in exosomes.

[0079] S3: The CD47 immune escape domain sequence and the CD63 exosome enrichment domain sequence were cloned a second time to obtain the chimeric molecule CD47-CD63.

[0080] like Figure 3 As shown, Figure 3 The image shows the electrophoresis sequence of the chimeric molecule CD47-CD63. The CD47 immune escape domain sequence is 423 base pairs from the N-terminus, and the CD63 exosome enrichment domain sequence is 561 base pairs from the C-terminus. The successfully assembled chimeric molecule CD47-CD63 consists of 984 base pairs. The electrophoretic band is located approximately 1 kb from the DNA ladder. Figure 3 The area selected by the black box in the middle;

[0081] The full-length cDNA sequence of CD47-CD63 with the Flag tag is as follows:

[0082]

[0083] The amino acid sequence of the CD47-CD63 protein with the flag tag is as follows:

[0084] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNESLLPVVIIAVGVFLFLVAFVGCCGACKE NYCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMDYKDDDDK.

[0085] S4: The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 is packaged with viruses to obtain a lentivirus overexpressing the chimeric molecule CD47-CD63.

[0086] In this process, the CD47 full-length cDNA lentiviral expression plasmid vector was packaged with viruses to obtain CD47-overexpressing lentiviruses for comparative experiments.

[0087] S5: Human bronchial epithelial cells (HBE) and mouse embryonic fibroblasts (NIH / 3T3) were infected with lentiviruses that overexpressed the chimeric molecule CD47-CD63, respectively. Stable CD47-CD63 protein overexpressing cell lines were obtained by screening with Puromycin antibiotic.

[0088] In this study, the CD47 lentivirus was used to infect human bronchial epithelial cells (HBE cells) and mouse embryonic fibroblasts (NIH / 3T3 cells), respectively. After screening with Puromycin antibiotic, stable CD47 overexpressing cell lines were obtained for comparative experiments.

[0089] Furthermore, after five or more consecutive passages, HBE cells were collected for Western blotting experiments. Control cells were cells lacking CD47 or CD47-CD63 protein. The expression levels of CD47-CD63 and CD47 were detected. Figure 4 It can be seen that CD47-CD63 and CD47 are stably overexpressed in HBE cells.

[0090] Furthermore, after five or more consecutive passages, NIH / 3T3 cells were collected for Western blotting experiments. Control cells were those lacking CD47 or CD47-CD63 protein. The expression levels of CD47-CD63 and CD47 were detected. Figure 5 It can be seen that CD47-CD63 and CD47 molecules are stably overexpressed in NIH / 3T3 cells.

[0091] S6: The CD47-CD63 protein stably overexpressing cell line is subjected to serum-free cell culture to obtain cell culture supernatant. The cell culture supernatant is then subjected to differential centrifugation and ultracentrifugation to obtain immune escape exosomes.

[0092] CD47 was also tested according to step S6 to obtain exosomes containing CD47.

[0093] Furthermore, after the stable CD47-CD63 overexpressing cell lines and the stable CD47 overexpressing cell lines were expanded and cultured in basal medium until the cell density was approximately 80%, the cells were washed with PBS and cultured in serum-free medium for 36 hours. The cell culture supernatant was collected and exosomes were separated by differential centrifugation at 300g for 10 minutes, 2000g for 10 minutes, 5000g for 30 minutes, and 10000g for 30 minutes, ultrafiltration through a 200 nm pore size membrane, and ultracentrifugation at 100000g for 90 minutes. After washing the exosomes with PBS, the exosomes were purified by ultracentrifugation at 100000g for 90 minutes to obtain a high-concentration exosome suspension enriched with CD47 protein and an exosome suspension of the chimeric molecule CD47-CD63 protein resuspended in physiological saline.

[0094] According to the method for preparing immune escape exosomes provided by the present invention, the steps of step S2 are as follows:

[0095] S21: Using the full-length cDNA of CD47 as a template, PCR cloning and amplification were performed using primers specific to the CD47 immune escape domain sequence containing the CD63 adapter sequence to obtain linear DNA encoding the CD47 immune escape domain.

[0096] S22: Using the full-length cDNA of CD63 as a template, PCR cloning and amplification were performed using primers specific to the CD63 exosome enrichment domain sequence with CD47 adapter sequence to obtain linear DNA encoding the CD63 exosome enrichment domain.

[0097] According to the method for preparing immune escape exosomes provided by the present invention, the steps of step S3 are as follows:

[0098] S31: Using the CD47 immune escape domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD47 pre-primer to obtain the first linear DNA.

[0099] S32: Using the CD63 exosome enrichment domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD63 back primer to obtain the second linear DNA.

[0100] S33: The first linear DNA and the second linear DNA are spliced ​​together to obtain the linear DNA of the chimeric molecule CD47-CD63.

[0101] According to the method for preparing immune escape exosomes provided by the present invention, step S4 is as follows:

[0102] The linear DNA of the chimeric molecule CD47-CD63 was digested and ligated with enzymes, and then inserted into the lentiviral expression vector pLV-EF1α-MCS-IRES Puro to obtain a lentiviral expression plasmid vector of the chimeric molecule CD47-CD63. The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 and the packaging plasmid were co-transfected into 293T cells to obtain lentivirus overexpressing the chimeric molecule CD47-CD63.

[0103] According to the method for preparing immune escape exosomes provided by the present invention, step S5 is as follows:

[0104] Lentiviral cells overexpressing the chimeric molecule CD47-CD63 were mixed with antibiotic-free medium at a 1:1 ratio to obtain a mixed medium. Polybrane was added to the mixed medium to culture HBE and NIH / 3T3 cells. After 12 hours, HBE and NIH / 3T3 cells were placed in basal medium and cultured for another 48 hours. Then, puro-resistant medium was added to the basal medium to screen and culture HBE and NIH / 3T3 cells to obtain a stable CD47-CD63 protein overexpressing cell line.

[0105] According to the present invention, a method for preparing immune escape exosomes is provided, wherein the basal culture medium is a culture medium supplemented with 1% antibiotics and 10% fetal bovine serum.

[0106] According to the method for preparing immune escape exosomes provided by the present invention, step S6 is as follows:

[0107] S61: The HBE and NIH / 3T3 cell lines that stably overexpress the CD47-CD63 protein were cultured in basal medium until the cell density reached approximately 80%.

[0108] S62: Wash the HBE and NIH / 3T3 cells, which were at a density of approximately 80%, with PBS, then add serum-free medium to the washed cells and culture for 36 hours.

[0109] S63: Collect the cell culture supernatant after culturing in the serum-free medium for 36 hours, centrifuge the cell culture supernatant at differential speed, then ultrafilter it with a nanoporous membrane, and finally separate and purify the exosomes in the cell culture supernatant by ultracentrifugation to obtain immune escape exosomes.

[0110] An immune escape exosome prepared according to a method for preparing immune escape exosomes provided by the present invention contains chimeric molecules CD47-CD63.

[0111] Exosomes were identified using electron microscopy, based on Figure 6 As can be seen, from left to right, they are exosomes derived from wild-type HBE cells, exosomes derived from HBE cells overexpressing CD47, and exosomes derived from HBE cells with the chimeric molecule CD47-CD63. Under an electron microscope, they all exhibit round or elliptical cup-shaped structures with a diameter of 50-200 nanometers, consistent with previously reported exosome structures. Figure 7 As can be seen from left to right, they are exosomes derived from wild-type NIH / 3T3 cells, exosomes derived from NIH / 3T3 cells overexpressing CD47, and exosomes derived from NIH / 3T3 cells with the chimeric molecule CD47-CD63. Under an electron microscope, they all appear as round or elliptical cup-shaped structures with a diameter of 50-200 nanometers, which is consistent with the exosome structures reported in the prior art.

[0112] To demonstrate that the exosome provided by this invention has excellent immune escape function, functional tests were conducted using Examples 1-8:

[0113] Example 1: Establishing an in vitro highly efficient immune clearance model of M1 macrophages using LPS-induced Raw 264.7 cells:

[0114] Raw 264.7 cells are murine monocytes / macrophages with some immune clearance capacity. Through LPS induction, they are activated into M1-type macrophages, exhibiting highly efficient immune clearance. To further verify the immune escape function of exosomes enriched with the chimeric molecule CD47-CD63 protein and enhance the phagocytic capacity of monocytes / macrophages, Raw 264.7 cells were induced into M1-type macrophages using LPS. Figure 8 As shown, Western blot experiments revealed that macrophage molecular marker F4 / 80 was expressed in both the control and LPS-induced groups, while the expression of M1 macrophage molecular markers TNF-α and iNOS was significantly higher in the LPS-induced group than in the control group.

[0115] according to Figure 9 As shown, immunofluorescence assays revealed that the macrophage molecular marker F4 / 80 was expressed in both the control and LPS-induced groups (displaying green fluorescence). The secondary antibody with green fluorescence 488 recognized the primary antibody F4 / 80. The expression of M1 macrophage molecular markers TNF-α and iNOS was significantly increased in the LPS-induced group, and the secondary antibody with red fluorescence 594 recognized the primary antibodies TNF-α and iNOS. Western blotting and immunofluorescence assays indicated that Raw 264.7 macrophages successfully differentiated into M1 macrophages under LPS induction.

[0116] Example 2: An experiment in which HBE cells overexpressing CD47 and HBE cells expressing the chimeric molecule CD47-CD63 evaded the immune clearance of M1 macrophages:

[0117] In vitro phagocytosis experiments were performed on wild-type HBE cells that overexpressed CD47 and the chimeric molecule CD47-CD63. M1 macrophages and HBE cells were stained with live cell dyes DiI and DiO, respectively. DiI staining made the membrane of M1 macrophages appear red (M1 macrophages / DiI in the figure), while DiO staining made HBE cells appear green (HBE cells / DiO in the figure). Co-incubation involved adding stained HBE cells to M1 macrophages at a ratio of 5:1. After co-incubation at 37°C for 4 hours, the cells were washed three times with PBS to remove excess unphagocytosed HBE cells. The two cell types exhibited different fluorescence, and phagocytosis was analyzed using a fluorescence microscope.

[0118] according to Figure 10 As can be seen from fluorescence microscopy, the phagocytosis of HBE cells by M1 macrophages is observed. M1 macrophages are red, and HBE cells are green. When the red and green channels are merged under the same microscope field, green cells can be observed to be localized within red cells, appearing yellow, confirming that HBE cells are phagocytosed by M1 macrophages. Comparing the phagocytosis of wild-type, CD47-overexpressing, and CD47-CD63-chimeric HBE cells by M1 macrophages, significant green fluorescence signals of HBE cells are visible in the red macrophages of the wild-type group, and significant yellow fluorescence co-localization signals are observed after merging. The green fluorescence signals of HBE cells in the red macrophages of the CD47-overexpressing group and the CD47-CD63-chimeric group are significantly reduced, and the yellow fluorescence co-localization signals are not obvious after merging.

[0119] The green fluorescence intensity of HBE cells and the red fluorescence intensity of macrophages were analyzed using ImageJ software. The red fluorescence intensity of macrophages from different groups was homogenized, and the green fluorescence intensity of different groups was used to quantify the amount of HBE cells phagocytosed. Figure 11 It can be seen that the number of HBE cells phagocytosed by the wild-type (HBE) group was significantly higher than that of the CD47 overexpression group (HBE-CD47) and the chimeric molecule CD47-CD63 group (HBE-CD47-CD63).

[0120] Example 3: NIH / 3T3 cells overexpressing CD47 and the chimeric molecule CD47-CD63 evade immune clearance by M1 macrophages.

[0121] In vitro phagocytosis experiments were performed on wild-type NIH / 3T3 cells that overexpressed CD47 and the chimeric molecule CD47-CD63. M1 macrophages and NIH / 3T3 cells were stained with live-cell dyes DiI and DiO, respectively. DiI staining made the membrane of M1 macrophages appear red (M1 macrophage / DiI in the figure), while DiO staining made NIH / 3T3 cells appear green (NIH / 3T3 cells / DiO in the figure). Co-incubation involved adding stained NIH / 3T3 cells to M1 macrophages at a ratio of 5:1. After co-incubation at 37°C for 4 hours, the cells were washed three times with PBS to remove excess unphagocytosed NIH / 3T3 cells. The two cell types exhibited different fluorescence, and phagocytosis was analyzed using a fluorescence microscope.

[0122] according to Figure 12 As can be seen from fluorescence microscopy, the phagocytosis of NIH / 3T3 cells by M1 macrophages was observed. M1 macrophages appeared red, while NIH / 3T3 cells appeared green. When the red and green channels were merged under the same microscope field, green cells were observed to be localized within red cells, appearing yellow, confirming that NIH / 3T3 cells were phagocytosed by M1 macrophages. Comparing the phagocytosis of wild-type, CD47-overexpressing, and CD47-CD63-overexpressing NIH / 3T3 cells by M1 macrophages, significant green fluorescence signals of NIH / 3T3 cells were observed in the red macrophages of the wild-type group, and significant yellow fluorescence co-localization signals were observed after merging. In the CD47-overexpressing and CD47-CD63-overexpressing groups, the green fluorescence signals of NIH / 3T3 cells in the red macrophages were significantly reduced, and the yellow fluorescence co-localization signals were not obvious after merging.

[0123] The green fluorescence intensity of NIH / 3T3 cells and the red fluorescence intensity of macrophages were analyzed using ImageJ software. The red fluorescence intensity of macrophages from different groups was homogenized, and the green fluorescence intensity of different groups was quantified to represent the amount of NIH / 3T3 cells phagocytosed. Figure 13It can be seen that the amount of NIH / 3T3 cells engulfed by the wild-type group (NH / 3T3) was significantly higher than that engulfed by the CD47 overexpression group (NH / 3T3-CD47) and the chimeric molecule CD47-CD63 group (NH / 3T3-CD47-CD63).

[0124] Example 4: Enrichment of the chimeric molecule CD47-CD63 protein in exosomes:

[0125] To verify the enrichment of overexpressed proteins in exosomes, the culture supernatants of wild-type HBE cells, cells overexpressing CD47 and the chimeric molecule CD47-CD63, and the culture supernatants of wild-type NIH / 3T3 cells, cells overexpressing CD47 and the chimeric molecule CD47-CD63, were collected. The exosomes were purified, and the enrichment of proteins in the exosomes was analyzed by Western blotting.

[0126] Alix and CD63 are exosomal molecular markers. Overexpression of CD47 and the chimeric molecule CD47-CD63 both carry protein tags. Figure 14 It can be seen that HBE cell-derived exosomes with wild type (Exo), CD47 overexpression (CD47-Exo), and chimeric molecule CD47-CD63 (CD47-CD63-Exo) are enriched with exosome markers Alix and CD63. CD63 is significantly increased due to the overexpression of chimeric molecule CD47-CD63. The Flag signals representing CD47 and chimeric molecule CD47-CD63 are significantly increased in CD47 and CD47-CD63, respectively, and are even higher in CD47-CD63 group, indicating that CD47-CD63 is significantly enriched in exosomes.

[0127] according to Figure 15 It can be seen that NIH / 3T3 cell-derived exosomes from wild-type (Exo), CD47-overexpressing (CD47-Exo), and chimeric molecule CD47-CD63 (CD47-CD63-Exo) are enriched with exosome markers Alix and CD63. CD63 is significantly increased due to the overexpression of the chimeric molecule CD47-CD63 group. The Flag signals representing CD47 and the chimeric molecule CD47-CD63 are significantly increased in CD47 and CD47-CD63, respectively, and are even higher in the CD47-CD63 group, indicating that CD47-CD63 is significantly enriched in exosomes.

[0128] Example 5: Enrichment of chimeric molecule CD47-CD63 exosomes to inhibit M1 macrophage phagocytosis:

[0129] To further verify the ability of CD47-CD63-enriched exosomes to evade immune clearance, exosomes were co-incubated with M1 macrophages, and the phagocytosis of exosomes was observed using a fluorescence microscope. Exosomes derived from wild-type HBE cells, those overexpressing CD47, and those derived from the CD47-CD63 chimeric molecule, as well as those derived from wild-type NIH / 3T3 cells, were stained with DiI (10 μM). When the M1 macrophage density in 24-well plates reached 50%, 0.2 μg of DiI-stained exosomes were added to each well. After co-incubation at 37°C for 15 minutes, the plates were washed three times with PBS to remove excess unphagocytosed exosomes. The phagocytosis of exosomes by M1 macrophages was then observed using a fluorescence microscope.

[0130] according to Figure 16 The study showed the phagocytosis of HBE cell-derived exosomes from wild-type (HBE-Exo), CD47-overexpressing (HBE-CD47-Exo), and the chimeric molecule CD47-CD63 (HBE-CD47-CD63-Exo) by M1 macrophages. Exosomes exhibited red fluorescence, while macrophage nuclei showed blue fluorescence. Significant red fluorescence was observed in the wild-type group, while the red fluorescence signal was significantly reduced in both the CD47 and CD47-CD63 groups, indicating that CD47-CD63 enrichment significantly promotes exosome immune escape. "Integration" refers to the addition of stained exosomes to macrophages, based on... Figure 16 It can be seen that the exosomes of the CD47-CD63 group have significant exosome immune escape function.

[0131] The red fluorescence intensity of exosomes and the blue fluorescence intensity of macrophage nuclei were analyzed using ImageJ software. The blue fluorescence intensity of macrophages from different groups was homogenized, and the red fluorescence intensity of different groups was quantified to represent the amount of HBE exosomes phagocytosed. Figure 17 It can be seen that the amount of exosomes phagocytosed by the wild-type group (HBE-Exo) was significantly higher than that of the CD47 overexpression group (HBE-CD47-Exo) and the chimeric molecule CD47-CD63 group (HBE-CD47-CD63-Exo).

[0132] according to Figure 18It can be seen that M1 macrophages exhibit significant phagocytosis of exosomes derived from wild-type (NIH / 3T3-Exo), CD47-overexpressing (NIH / 3T3-CD47-Exo), and chimeric molecule CD47-CD63 (NIH / 3T3-CD47-CD63-Exo) NIH / 3T3 cells. The wild-type group showed significant red fluorescence, while the CD47 group and CD47-CD63 group showed significantly reduced red fluorescence, indicating that enrichment of CD47-CD63 can significantly promote exosome immune escape. "Integration" refers to the addition of stained exosomes to macrophages, based on... Figure 18 It can be seen that the exosomes of the CD47-CD63 group have significant exosome immune escape function.

[0133] The red fluorescence intensity of exosomes and the blue fluorescence intensity of macrophage nuclei were analyzed using ImageJ software. The blue fluorescence intensity of macrophages from different groups was homogenized, and the red fluorescence intensity of different groups was quantified to represent the amount of NIH / 3T3 exosomes phagocytosed. Figure 19 It can be seen that the amount of exosomes phagocytosed by the wild-type group (NIH / 3T3-Exo) was significantly higher than that of the CD47 overexpression group (NIH / 3T3-CD47-Exo) and the chimeric molecule CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo).

[0134] Example 6: Single-particle tracking experiment to analyze the diffusion and phagocytosis of exosomes on the surface of M1 macrophages:

[0135] To further analyze the process of CD47-CD63-enriched chimeric exosomes escaping phagocytosis by M1 macrophages, a single-particle tracking assay was used to analyze the diffusion trajectory of individual exosomes on the surface of M1 macrophages and the time it took for individual exosomes to be phagocytosed by M1 macrophages. Exosomes derived from HBE and NIH / 3T3 cells were stained (DiI, 10 μM), and images of the phagocytic process were acquired using a research-grade inverted biological microscope. 50 μg of exosomes were added to a confocal culture dish of M1 macrophages, and images were immediately acquired using dual-channel imaging (bright field and red excitation channel) at 1 frame / 10 s. After acquisition, the enrichment of exosomes within macrophages was recorded, and the phagocytic process of M1 macrophages phagocytosing different groups of exosomes was analyzed. The diffusion trajectory of individual exosomes on the macrophage surface was plotted using the exosome location coordinates from the acquired images.

[0136] according to Figure 20It can be seen that M1 macrophages phagocytose exosomes derived from HBE cells, including wild-type (HBE-Exo), CD47-overexpressing (HBE-CD47-Exo), and the chimeric molecule CD47-CD63 (HBE-CD47-CD63-Exo). In the wild-type group, a single exosome indicated by the arrow after 4 consecutive images was phagocytosed by M1 macrophages. In the CD47 group, a single exosome indicated by the arrow after 30 consecutive images was not phagocytosed by M1 macrophages. In the CD47-CD63 group, a single exosome indicated by the arrow after 100 consecutive images was not phagocytosed by M1 macrophages.

[0137] Furthermore, according to Figure 21 As can be seen from left to right, the diffusion trajectories of HBE cell-derived exosomes—a single wild-type exosome, one overexpressing CD47, and one expressing the chimeric molecule CD47-CD63—on the surface of M1 macrophages show the following patterns: wild-type exosomes were phagocytosed after a few diffusions; CD47-overexpressing exosomes diffused multiple times on the cell surface but were partially phagocytosed; and CD47-CD63-overexpressing exosomes diffused on the cell surface for a prolonged period without being phagocytosed.

[0138] according to Figure 22 It can be seen that M1 macrophages phagocytose exosomes derived from NIH / 3T3 cells, including wild-type (NIH / 3T3-Exo), CD47-overexpressing (NIH / 3T3-CD47-Exo), and the chimeric molecule CD47-CD63 (NIH / 3T3-CD47-CD63-Exo). In the wild-type group, a single exosome, indicated by the arrow, is phagocytosed by M1 macrophages after three consecutive images. In the CD47 group, a single exosome, indicated by the arrow, is phagocytosed by M1 macrophages after 30 consecutive images. In the CD47-CD63 group, a single exosome, indicated by the arrow, is not phagocytosed by M1 macrophages after 100 consecutive images. Figure 23 As can be seen from left to right, these are the diffusion trajectories of NIH / 3T3 cell-derived exosomes, single wild-type exosomes, exosomes overexpressing CD47, and exosomes overexpressing the chimeric molecule CD47-CD63, on the surface of M1 macrophages. The trajectories show that wild-type exosomes were phagocytosed after a few diffusions, exosomes overexpressing CD47 were partially phagocytosed after multiple diffusions on the cell surface, and exosomes overexpressing the chimeric molecule CD47-CD63 diffused on the cell surface for a long time without being phagocytosed.

[0139] To further analyze the efficiency of exosomes escaping M1 macrophages, the phagocytic time of exosomes from NIH / 3T3 cells under multiple single-particle tracking was statistically analyzed. As shown in Table 1, the phagocytic time of 10 exosomes from wild-type (NIH / 3T3-WT-Exo), CD47-overexpressing (NIH / 3T3-CD47-Exo), and the chimeric molecule CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo) were statistically analyzed. The average time was 33 seconds for the wild-type group, 309 seconds for the CD47 group, and 1019 seconds for the CD47-CD63 group.

[0140] Table 1. Statistics on phagocytic time of exosomes derived from NIH / 3T3 cells

[0141]

[0142] Example 7: Enrichment of chimeric CD47-CD63 exosomes inhibits phagocytic function of circulatory mononuclear cells:

[0143] To further verify the ability of CD47-CD63-enriched chimeric exosomes to evade immune clearance by circulatory monocytes, an in vitro blood cell phagocytosis experiment was conducted. Monocytes, including neutrophils and macrophages, are cells with phagocytic activity in the circulatory system. The experiment aimed to verify the inhibitory effect of CD47-CD63-enriched chimeric exosomes on monocyte phagocytosis in the blood. Using an in vitro method simulating exosome entry into the circulatory system, 100 μL of fresh blood was collected from the retro-orbital vein of mice, and 6 μg of DiI-stained (10 μM) exosomes were added. After mixing thoroughly and incubating at room temperature for 15 minutes, blood smears were prepared. The phagocytosis of exosomes by monocytes was observed and statistically analyzed using confocal fluorescence microscopy. Figure 24 As can be seen from left to right, the images show the accumulation of exosomes in circulatory system monocytes 15 minutes after phagocytosis of exosomes from wild-type, CD47-overexpressing, and CD47-CD63-overexpressing NIH / 3T3 cells. In the wild-type group, the blue cell nuclei are almost purple due to the strong red signal, while the red signal is weak in the CD47 and CD47-CD63 groups, resulting in blue cell nuclei. Furthermore, a large number of red fluorescent exosomes are visible in the wild-type group monocytes, while the number of red fluorescent exosomes is significantly reduced in the CD47 and CD47-CD63 group monocytes.

[0144] The intensity of red fluorescence of exosomes and the number of circulatory system monocytes were analyzed using ImageJ software. The number of circulatory system monocytes was homogenized across different groups, and the red fluorescence intensity of different groups was quantified to represent the amount of phagocytosed exosomes. Figure 25It can be seen that after 15 minutes of phagocytosis by circulatory system monocytes, the wild-type group (NIH / 3T3-Exo) phagocytosed a large number of exosomes, while the phagocytosis of exosomes by the CD47 group (NIH / 3T3-CD47-Exo) and the chimeric molecule CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo) was significantly reduced.

[0145] Example 8: In vivo mouse experiments to verify the immune escape function of enriched chimeric molecule CD47-CD63 exosomal:

[0146] To further verify the immune escape function of enriched chimeric molecule CD47-CD63 exosomes, in vivo experiments were conducted in mice to verify the circulating half-life and tissue distribution of exosomes. NIH / 3T3 cell-derived exosomes were used as the experimental material in mice, and experiments were conducted in 6-week-old male ICR mice (average weight 32-38g). The mice were divided into four groups of four, each receiving an injection of 150 μg of fluorescently labeled DiR-stained exosomes. 100 μL of blood was collected from the retro-orbital vein at different time points: 2 minutes, 10 minutes, and 60 minutes after injection. After coagulation, serum was collected by centrifugation, and the residual amount of exosomes in the serum was analyzed at different time points. After the last blood collection, the mice were euthanized by cervical dislocation, and major organs were harvested for in vitro fluorescence imaging. The fluorescence intensity of aggregated exosomes in each organ was analyzed, and the enrichment of exosomes in mouse organs was compared among the groups.

[0147] The residual exosomes in serum 10 minutes after exosome injection were analyzed using lipid capture exosome technology. The positive control group was serum with 6 μg of exosomes added directly in vitro, and the negative control group was serum from control mice. The residual exosomes in serum were observed using a dark-field microscope at 100x oil immersion.

[0148] according to Figure 26 As can be seen from left to right, the groups are the negative control group, wild type (NIH / 3T3-Exo), CD47 overexpression group (NIH / 3T3-CD47-Exo), chimeric CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo), and positive control group. Ten minutes after exosome injection, the wild type group had very few green fluorescent exosomes remaining in its serum, the CD47 group had a small amount of green fluorescent exosomes remaining in its serum, and the CD47-CD63 group had a large amount of green fluorescent exosomes remaining in its serum. This indicates that enriching CD47-CD63 exosomes can significantly evade clearance by the immune system.

[0149] Exosome counts were performed on serum exosome residues observed under dark-field microscopy. Ten fields of view were randomly selected from each group, and the number of green fluorescent dots was counted as the number of residual exosomes. Figure 27It can be seen that the wild-type group (NIH / 3T3-Exo) had an average of 4 residual exosomes per field of view, the CD47 group (NIH / 3T3-CD47-Exo) had an average of 11 residual exosomes per field of view, and the CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo) had an average of 33 residual exosomes per field of view, further demonstrating that enrichment of CD47-CD63 exosomes can significantly evade clearance by the immune system.

[0150] The distribution of red-fluorescent exosomes in mouse organs and tissues was analyzed using in vitro organ imaging technology. Figure 28 It can be seen that exosomes mainly accumulate in the liver, lungs, and spleen, while almost no exosomes accumulate in the heart and kidneys. In the wild-type group (NIH / 3T3-Exo), the liver, lungs, and spleen showed significant red fluorescence intensity, with a large amount of exosome accumulation; in the CD47 group (NIH / 3T3-CD47-Exo), the liver, lungs, and spleen showed strong red fluorescence intensity, with a relatively large amount of exosome accumulation; in the CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo), the red fluorescence intensity in the liver and lungs was significantly reduced, and the spleen showed almost no red fluorescence intensity, with a small amount of exosomes accumulating in the liver and lungs. This indicates that enriching CD47-CD63 exosomes can effectively evade clearance by the immune system.

[0151] The intensity of red fluorescence in in vitro tissues and organs was analyzed using ImageJ software. Quantitative analysis was performed on exosomes with red fluorescence aggregated in each group of organs. Figure 29 As can be seen, quantifying the fluorescence intensity in the liver showed that there was no significant difference in the red fluorescence intensity of the liver between the wild-type group (NIH / 3T3-Exo) and the CD47 group (NIH / 3T3-CD47-Exo). Compared with the wild-type group, the CD47-CD63 group (NIH / 3T3-CD47-CD63-Exo) showed a significant decrease in the intensity of accumulated red fluorescence in the liver, indicating that enrichment of CD47-CD63 can significantly reduce the immune clearance of exosomes from the liver.

[0152] In summary, in vitro phagocytosis experiments by macrophages and in vivo serum exosome residue and organome distribution experiments verified the ability of exosomes to evade immune clearance. The results showed that cells overexpressing the chimeric molecule CD47-CD63 and exosomes enriched with the chimeric molecule CD47-CD63 both inhibited phagocytosis by phagocytes and effectively increased the circulating half-life of exosomes in vivo. Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the present invention are all within the scope of protection claimed by the present invention.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing immune-escape exosomes, characterized in that, Includes the following steps: S1: Based on the CD47 and CD63 gene CDS sequences, using HBE cell cDNA as a template, the full-length cDNA of CD47 and CD63 genes was cloned by PCR to obtain the full-length cDNA of CD47 and CD63. S2: Based on the full-length cDNA of CD47 and CD63, the sequence of the CD47 immune escape domain and the sequence of the CD63 exosome enrichment domain were determined. S3: The CD47 immune escape domain sequence and the CD63 exosome enrichment domain sequence were cloned a second time to obtain the chimeric molecule CD47-CD63. S4: The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 is packaged with viruses to obtain a lentivirus overexpressing the chimeric molecule CD47-CD63. S5: Human bronchial epithelial cells (HBE) and mouse fibroblasts (NIH / 3T3) were infected with lentiviruses that overexpressed the chimeric molecule CD47-CD63 to obtain stable CD47-CD63 overexpressing cell lines. S6: The CD47-CD63 stably overexpressing cell line was cultured in serum-free medium to obtain cell culture supernatant. The cell culture supernatant was then centrifuged differentially and then ultracentrifuged to obtain immune-escape exosomes.

2. The method for preparing immune-escape exosomes according to claim 1, characterized in that, The steps for step S2 are as follows: S21: Using the full-length cDNA of CD47 as a template, PCR cloning and amplification were performed using primers specific to the CD47 immune escape domain sequence containing the CD63 adapter sequence to obtain linear DNA encoding the CD47 immune escape domain. S22: Using the full-length cDNA of CD63 as a template, PCR cloning and amplification were performed using primers specific to the CD63 exosome enrichment domain sequence with CD47 adapter sequence to obtain linear DNA encoding the CD63 exosome enrichment domain.

3. The method for preparing immune-escape exosomes according to claim 2, characterized in that, The steps for step S3 are as follows: S31: Using the CD47 immune escape domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD47 pre-primer to obtain the first linear DNA. S32: Using the CD63 exosome enrichment domain-encoded linear DNA as a template, PCR cloning and amplification were performed using the CD63 back primer to obtain the second linear DNA. S33: The first linear DNA and the second linear DNA are spliced ​​together to obtain the linear DNA of the chimeric molecule CD47-CD63.

4. The method for preparing immune-escape exosomes according to claim 3, characterized in that, The steps for step S4 are as follows: The linear DNA of the chimeric molecule CD47-CD63 was digested and ligated with enzymes, and then inserted into the lentiviral expression vector pLV-EF1α-MCS-IRES Puro to obtain a lentiviral expression plasmid vector of the chimeric molecule CD47-CD63. The lentiviral expression plasmid vector of the chimeric molecule CD47-CD63 and the packaging plasmid were co-transfected into 293T cells to obtain lentivirus overexpressing the chimeric molecule CD47-CD63.

5. The method for preparing immune-escape exosomes according to claim 1, characterized in that, The steps for step S5 are as follows: Lentiviral cells overexpressing the chimeric molecule CD47-CD63 were mixed with antibiotic-free medium at a 1:1 ratio to obtain a mixed medium. Polybrane was added to the mixed medium to culture HBE and NIH / 3T3 cells. After 12 hours, HBE and NIH / 3T3 cells were placed in basal medium and cultured for another 48 hours. Then, puro-resistant medium was added to the basal medium to screen and culture HBE and NIH / 3T3 cells to obtain a stable CD47-CD63 protein overexpressing cell line.

6. The method for preparing immune-escape exosomes according to claim 5, characterized in that, The basal culture medium is a culture medium supplemented with 1% antibiotics and 10% fetal bovine serum.

7. The method for preparing immune-escape exosomes according to claim 1, characterized in that, The steps for step S6 are as follows: S61: The HBE and NIH / 3T3 cell lines stably overexpressing the CD47-CD63 protein were cultured in basal medium until the cell density reached 80%. S62: Wash the HBE and NIH / 3T3 cells at a density of 80% with PBS, then add serum-free medium to the washed cells and culture for 36 hours; S63: Collect the cell culture supernatant after 36 hours, centrifuge the cell culture supernatant at differential speed, then ultrafilter it with a nanoporous membrane, and finally separate and purify the exosomes in the cell culture supernatant by ultracentrifugation to obtain immune escape exosomes.

8. An immune escape exosome prepared according to the method for preparing immune escape exosomes according to any one of claims 1-7, characterized in that, It contains chimeric molecules CD47-CD63.

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