Exosome production and use

Abstract

To provide a method for promoting the production of exosomes.SOLUTION: The present invention provides a method for producing exosomes on a large scale by using a cyclic tensile bioreactor to stimulate cells to release exosomes. In addition, the present invention also provides an exosome having an anti-HLA-G protein specific for cancer, and the exosome is used as a delivery vehicle to deliver therapeutic agents for treating cancer.SELECTED DRAWING: Figure 17

Description

[Technical Field]

[0001] This invention relates to a method for large-scale exosome production using a periodic tension bioreactor that stimulates cells to release exosomes. Furthermore, this invention relates to exosomes possessing cancer-specific anti-HLA-G proteins. [Background technology]

[0002] In recent years, various pharmaceutical companies have been investing significant resources in the development of novel drug carriers that are expected to deliver small molecule drugs or biologics more efficiently and accurately. Exosomes are considered excellent candidates for drug carriers because they have low immunogenicity, high biocompatibility, high biological activity, low cellular rejection, and are nano-sized relative to the microenvironment. In addition to these conventional advantages, exosomes possess other superior properties, such as high affinity, ease of phagocytosis by target cells, ease of intracellular drug degradation and release, and avoidance of consumption by the immune system. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] The present invention provides a method for promoting exosome production, the method comprising (a) providing a periodic tension bioreactor, the periodic tension bioreactor comprising a culture chamber and a growth stimulation promoting structure Biocompatible polymer materials It comprises multiple tensile components, Biocompatible polymer materials And multiple tensile components are placed inside the culture chamber. Biocompatible polymer materials It includes exosome-producing cells, Biocompatible polymer materials (b) providing a biocompatible polymer material by repeatedly stretching a biocompatible polymer material by applying a periodic tensile force using the multiple tensile components, and (c) recovering exosomes released from exosome-producing cells by the periodic tensile force applied by the multiple tensile components. [Means for solving the problem]

[0004] The present invention also provides an exosome comprising a fusion protein, wherein the fusion protein comprises a target protein and an exosome transmembrane protein, and the target protein comprises an anti-HLA-G protein. The present invention further provides a method for treating a subject having cancer, comprising administering a composition to the subject having cancer, wherein the composition comprises a therapeutic exosome, and the therapeutic exosome comprises the above-mentioned fusion protein and an anticancer agent. [Brief explanation of the drawing]

[0005] [Figure 1A] This figure shows the results of morphological analysis of exosomes with and without cargo engineering technology. [Figure 1B] This figure shows the results of verifying the size of exosomes with and without cargo engineering technology. Exo: exosome; CARESO: cargo exosome. [Figure 2A] This figure shows the identification of exosome biomarkers (CD63, CD81, CD9, Alix, HSP-70, and β-tubulin) using West blot. [Figure 2B] This figure shows the identification of exosome biomarkers (CD63, CD81, and CD9) using flow cytometry. PE-A: Phycoerythrin region. [Figure 3] This figure shows the evaluation of exosome (green) retention in MDA-MB-231 cells (F-actin: red, nucleus: blue) using a confocal microscope. Exosome: green, F-actin: red, nucleus: blue. [Figure 4] This figure compares the effects of exosome uptake using fluorescent staining in breast cancer cells (MDA-MB-231) and normal mammary cells (MCF-10A), with and without targeting function. Exosomes: green, F-actin: red, nucleus: blue. [Figure 5]This figure compares the effects of exosome uptake using flow cytometry in breast cancer cells (MDA-MB-231) and normal mammary cells (MCF-10A), with and without targeting function. [Figure 6] This figure compares the effectiveness of target exosome uptake by breast cancer cells (MDA-MB-231) and normal mammary cells (MCF-10A) in a co-culture system using fluorescent staining. Exosomes: green, F-actin: red, nucleus: blue. HPF: human lung fibroblasts. [Figure 7] This figure shows the evaluation of the cytotoxicity of exosomes against breast cancer cells (MDA-MB-231) and normal mammary cells (MCF-10A), with and without targeting function. Ctl: control. [Figure 8] This figure shows the evaluation of the in vivo therapeutic ability of CARExo's in vivo distribution. In this invention, a xenograft mouse model is constructed using MD-MBA-231 cells. [Figure 9] This figure shows the amounts of miRNA and DOX co-incorporated into exosomes. DOX: Doxorubicin. [Figure 10] This figure shows the evaluation of the cytotoxicity of exosomes incorporating miRNA and DOX into MDA-MB-231. Ctl: control. [Figure 11] This figure shows the evaluation of the effect of miRNA-containing exosomes (miR-34a@CARExo) on intracellular miRNA release by fluorescent staining. [Figure 12A] This figure shows the results of the verification of the effectiveness of miRNA / DOX-containing CARExo in inhibiting breast cancer tumors in vivo. [Figure 12B] This figure shows the results of evaluating the effectiveness of miRNA / DOX-containing CARExo in tumor metastasis to other organs. Ctl: control. [Figure 13]This is a figure showing the comparison of the uptake effect of exosomes in MB11 (glioblastoma), A549 (non-small cell lung cancer), HepG2 (hepatocellular carcinoma), OECM (oral squamous cell carcinoma), SKOV3 (ovarian cancer cell line), and T24 (bladder cancer) with or without the target function by fluorescence staining. Exosomes: green, F-actin: red, nucleus: blue. [Figure 14] This is a figure showing the comparison of the cytotoxicity of DOX-containing exosomes in breast cancer (MB231), lung cancer (HCC827), oral cancer (OECM), liver cancer (HepG2), colon cancer (CaCO2), medulloblastoma (U87), bladder cancer (T24), pancreatic cancer (MIA PaCa-2), and ovarian adenocarcinoma (SKOV3) with or without the target function. Mammary epithelial cells (MePic) and human lung fibroblasts (HPF) are normal cells. [Figure 15] This is a figure showing the comparison of the cytotoxicity of exosomes containing various chemotherapeutic agents in breast cancer (MB231) with or without the target function. Ctl: control. [Figure 16A] This is a figure showing the preparation of cell-containing FGelMa hydrogel and the use of a cyclic stretching culture system. A scheme showing the procedure used to prepare the cell-containing growth-stimulating promoting hydrogel is shown. [Figure 16B] This is a figure showing the preparation of cell-containing FGelMa hydrogel and the use of a cyclic stretching culture system. A figure showing the as-prepared growth-stimulating promoting hydrogel immersed in the culture medium is shown. [Figure 16C] This is a figure showing the preparation of cell-containing FGelMa hydrogel and the use of a cyclic stretching culture system. A figure showing that the cell-containing growth-stimulating promoting hydrogel was applied to a dynamic tensile culture system and cultured under cyclic stretching at a strain of 20% and a frequency of 0.48 Hz is shown. [Figure 17] This is a figure showing the comparison of the yield of exosomes produced by a HEK293T-containing growth-stimulating promoting scaffold in a tensile culture system with the yield of exosomes secreted by single cells. [Figure 18] This is a figure showing the differences in cell morphology and YAP protein staining of a HEK293T-containing growth-stimulating promoting scaffold in a cyclic tensile culture system. [Figure 19A] This is a diagram showing the differences in cell morphology and YAP protein staining of a growth-stimulating promoting scaffold containing HEK293 using a YAP inhibitor (verteporfin) in a cyclic tensile culture system. [Figure 19B] This is a diagram showing the differences in exosome secretion of a growth-stimulating promoting scaffold containing HEK293 using a YAP inhibitor (verteporfin) in a cyclic tensile culture system. [Figure 20] This is a diagram showing the differences in the morphology, size, and biomarkers of HEK293-related exosomes between a static culture system and a dynamic culture system.

Mode for Carrying Out the Invention

[0006] The present invention establishes a high-quality and mass-production engineering technology-modified exosome platform by adjusting parent cell culture parameters. The parent cell (HEK293) secretes a large amount of high-purity exosomes as biological products through a 3D dynamic stimulation process. In the present invention, three platforms are formed, such as a gene-modified membrane targeting HLA-G, an exosome mass production system, and a combined drug uptake design. The present invention can achieve several results: 1) The amount of exosomes secreted from HEK293 under a dynamic culture environment can produce 45 to 300 times the exosomes having highly expressed CD63, thereby bringing high-quality and high-quantity exosomes for treatment. 2) Due to the low uptake of chemotherapeutic agents, cancer cells can be killed with high efficiency without side effects. 3) CARExos carries anti-HLA-G protein and miRNA 34a, thereby suppressing tumor progression and decelerating the tumor metastasis process. 4) CARExos shows excellent tumor size control in model animals and does not show metastasis. The present invention believes that CARExos has a high possibility of becoming an efficient anti-cancer weapon for patients in the future and has great potential and business opportunities.

[0007] As used herein, the terms “one” or “one” describe elements and components of the present invention. These terms are used solely for explanatory purposes and to convey the basic ideas of the invention. This description should be understood to include one or at least one, and unless otherwise clearly indicated, the singular form also includes the plural form. When used in the claims in combination with the word “comprising,” the term “one” may mean one or more.

[0008] As used in the claims herein, the term “or” means “and / or” unless expressly indicated otherwise, or unless the other options are mutually exclusive.

[0009] The present invention provides a method for promoting exosome production, the method comprising (a) providing a periodic tension bioreactor, the periodic tension bioreactor comprising a culture chamber and a growth stimulation promoting structure Biocompatible polymer materials It comprises multiple tensile components, Biocompatible polymer materials And multiple tensile components are placed inside the culture chamber. Biocompatible polymer materials It includes exosome-producing cells, Biocompatible polymer materials The ends of the device are connected by multiple tensile components; (b) repeatedly stretching a biocompatible polymer material by multiple tensile components to apply periodic tensile force; and (c) recovering exosomes released from exosome-producing cells by periodic tensile force applied by multiple tensile components.

[0010] In one embodiment, the biocompatible polymer material includes a methacryloyl polymer. In a preferred embodiment, the methacryloyl polymer includes gelatin methacryloyl (GelMa), collagen methacryloyl, hyaluronic acid methacryloyl (HAMa), chondroitin sulfate methacryloyl, chitosan methacryloyl, alginate methacryloyl, or a decellularized extracellular matrix (dECMMa) containing methacryloyl. In a more preferred embodiment, the gelatin methacryloyl includes fish gelatin methacryloyl (FGelMa) or porcine gelatin methacryloyl (PGelMa). Furthermore, the gelatin methacrylamide is prepared from gelatin derived from fish or pigs, methacrylic anhydride, and an optional solution (e.g., phosphate buffer solution). Thus, the methacrylamide polymer is biodegradable and biocompatible.

[0011] In this invention, the concentration of the biocompatible polymer material affects its growth-stimulating function, which in turn affects exosome production. Therefore, this invention uses biocompatible polymer materials having different concentrations to affect the exosome yield. For example, the concentration of GelMa is in the range of 5% to 30% by weight, the concentration of methacryloyl alginate is in the range of 1% to 10% by weight, the concentration of collagen methacryloyl is in the range of 0.5% to 10% by weight, the concentration of HAMA is in the range of 1% to 10% by weight, and the concentration of dECMMa is in the range of 1% to 10% by weight. In one embodiment, the concentration of the biocompatible polymer material is in the range of 0.1% to 50% by weight. In a preferred embodiment, the concentration of the biocompatible polymer material is in the range of 0.2% to 40% by weight. In a more preferred embodiment, the concentration of the biocompatible polymer material is in the range of 0.5% to 30% by weight.

[0012] In this invention, the concentration of the biocompatible polymer material is adjusted by dilution. For example, if the biocompatible polymer material is FGelMa, 20% by weight of FGelMa is prepared by mixing 20g of FGelMa with 80g of distilled water.

[0013] Furthermore, the biocompatible polymer material having the growth-stimulating structure can withstand at least 5% of the tensile strain applied by the tensile component without tearing. In one embodiment, the mechanical strength of the biocompatible polymer material having the growth-stimulating structure withstands a tensile strain of 2.5 to 50%. In a preferred embodiment, the mechanical strength of the biocompatible polymer material having the growth-stimulating structure withstands a tensile strain of 5 to 40%. In a more preferred embodiment, the mechanical strength of the biocompatible polymer material having the growth-stimulating structure withstands a tensile strain of 10 to 30%.

[0014] In some embodiments, the term “tensile strain” is defined as the deformation or elongation of a solid due to the application of a tensile force or stress. In other words, tensile strain occurs when an applied force attempts to stretch it, causing the length of the body to increase. Tensile strain can be mathematically expressed by the following equation: ε = ΔL / L, where ε = tensile strain, ΔL = change in length, and L = original length.

[0015] In this invention, the biocompatible polymer material is designed to have a dumbbell-like shape with wider ends than the main body. Thus, the biocompatible polymer material can be divided into two parts: one part is a first polymer material having a growth-stimulating structure, and the other part comprises two second polymer materials having a rigid structure. Therefore, the mechanical strength of each second polymer material having a rigid structure is higher than the mechanical strength of the first polymer material having a growth-stimulating structure. These two second polymer materials are each connected to both ends of the first polymer material. Cells are taken up by the growth-stimulating structure of the first polymer material. Thus, the first polymer material is used to take up cells, and the second polymer materials are used to provide appropriate connections between the first polymer material and multiple tensile components.

[0016] In one embodiment, the biocompatible polymer material comprises a first polymer material and two second polymer materials. In a preferred embodiment, these two second polymer materials are each connected to both ends of the first polymer material. In a more preferred embodiment, the first polymer material is used to take up exosome-producing cells, and the second polymer materials are used to connect the first polymer material to multiple tensile components. In another embodiment, the first polymer material is FGelMa, and the second polymer material is GelMa.

[0017] These exosome-producing cells can be incorporated into a biocompatible polymer material or a first polymer material by known methods. After the exosome-producing cells are incorporated into a biocompatible polymer material having a growth-stimulating structure, the present invention can obtain a cell-containing growth-stimulating scaffold. In the present invention, the biocompatible polymer material is cell-containing FgelMa, and the cell-containing FgelMa is obtained by mixing exosome-producing cells with FgelMa.

[0018] Exosome-producing cells can exist in the form of, for example, primary cells, cell lines, cells present in multicellular organisms, or essentially any other type of cell source and exosome-producing cell material. The term “exosome-producing cells” can be understood as relating to any type of cell capable of producing exosomes under favorable conditions, for example, in suspension culture or adherent culture or in any other type of culture system. Exosome-producing cells also include cells that produce exosomes in vivo. Exosome-producing cells can be selected from a wide range of cells and cell lines that can grow in suspension culture or adherent culture, or that can be adapted to suspension growth. Exosome-producing cells can be selected from a group including mesenchymal stem or stromal cells (which can be obtained from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, placenta, tooth buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells, and more specifically, amniotic epithelial cells, myelosuppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, etc., optionally expressing various early markers. Particularly targeted cell lines include endothelial cell lines such as human umbilical cord endothelial cells (HUVECs), human embryonic kidney (HEK) cells, microvascular or lymphatic endothelial cells, erythrocytes, erythrocyte progenitor cells, chondrocytes, mesenchymal stem cells (MSCs) of different origins, amniotic cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from the placenta, or epithelial cells, fibroblasts, and endothelial cells from the airways or alveoli. Immune cells such as B cells, T cells, NK cells, macrophages, monocytes, and dendritic cells (DCs) are also within the scope of this invention, and essentially any type of cell capable of producing exosomes is also included herein. In general, exosomes can originate from essentially any cell source, primary cell source, or immortalized cell line. Particularly targeted cell lines include human umbilical cord endothelial cells (HUVECs), human embryonic kidney (HEK) cells, such as HEK293 cells, HEK293 T cells, serum-free HEK293 cells, suspension HEK293 cells, endothelial cell lines, such as microvascular or lymphatic endothelial cells, erythrocytes, erythrocyte progenitor cells, chondrocytes, MSCs of different origins, amniotic cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, and epithelial cells.In one embodiment, the exosome-producing cells include mammalian cells. In a preferred embodiment, the exosome-producing cells include embryonic kidney cells. In a more preferred embodiment, the exosome-producing cells include HEK293 cells. Thus, the exosome-producing cells can produce exosomes that retain the biological properties of exosomes, and these exosomes may express CD9, CD63, CD81, and HSP70.

[0019] In the present invention, a plurality of pores are present in the biocompatible polymer material or the first polymer material. The plurality of pores are designed to enhance the growth-stimulating function of the growth-stimulating structure in the biocompatible polymer material or the first polymer material. In addition, the plurality of pores allow the culture medium to flow into the biocompatible polymer material or the first polymer material for cell culture. In one embodiment, the biocompatible polymer material or the first polymer material includes a plurality of pores. The plurality of pores can be of various types depending on the need for growth stimulation. In one embodiment, the shape of the pores includes spherical, ellipsoidal, rhomboid, or spindle-shaped. In a preferred embodiment, the shape of the pores is spindle-shaped. In another embodiment, the spindle-shaped pores are designed to have a length of 1 to 5 mm and a width of 0.1 to 0.5 mm. In a preferred embodiment, the spindle-shaped pores are designed to have a length of 2 to 4 mm and a width of 0.2 to 0.4 mm. Furthermore, multiple pores are designed to penetrate the structure of the biocompatible polymer material or the first polymer material.

[0020] In some embodiments, the culture chamber is filled with a culture medium, and the biocompatible polymer material is placed in the culture medium. In one embodiment, the culture chamber contains a culture medium.

[0021] In various embodiments, periodic tensile force is applied by multiple tensile components. Biocompatible polymer materials having a growth-stimulating structure are trained by periodic tensile force from multiple tensile components. In one embodiment, the periodic tensile force applied by multiple tensile components is a tensile strain of 2.5 to 50%. In a preferred embodiment, the periodic tensile force applied by multiple tensile components is a tensile strain of 5 to 40%. In a more preferred embodiment, the periodic tensile force applied by multiple tensile components is a tensile strain of 10 to 30%. In another embodiment, the periodic tensile force applied by multiple tensile components is at a frequency of 0.1 to 4 Hz. In a preferred embodiment, the periodic tensile force applied by multiple tensile components is at a frequency of 0.2 to 2 Hz. In a more preferred embodiment, the periodic tensile force applied by multiple tensile components is at a frequency of 0.3 to 1 Hz. In one embodiment, multiple tensile components impart periodic uniaxial tensile force to the biocompatible polymer material. In another embodiment, periodic tensile forces of multiple tensile components are applied horizontally to the biocompatible polymer material. Thus, the biocompatible polymer material containing exosome-producing cells is treated with periodic tensile forces to stimulate the cells to release exosomes.

[0022] In the present invention, a biocompatible polymer material is subjected to periodic tensile force for more than one week, and then exosomes are recovered from exosome-producing cells in the biocompatible polymer material. In one embodiment, the recovery of exosomes in step (c) is performed about one week, two weeks, three weeks, one month, two months, or three months after the periodic tensile force is applied. In a preferred embodiment, the recovery of exosomes in step (c) is performed about one to two months after the periodic tensile force is applied. In another embodiment, the number of exosomes recovered one month after the periodic force is applied is 0.1 x 10 per cell. 6 ~2x10 6 In a preferred embodiment, the number of exosomes recovered one month after the application of periodic force is 0.5 x 10 per cell. 6 ~1.5x10 6That is the case.

[0023] When periodic tensile force is applied to cells, the cells transform from single cells into self-organizing three-dimensional (3D) cell spheroids and express large amounts of Yes-related protein (YAP). This invention demonstrates that periodic tensile force can stimulate cells to express large amounts of YAP protein, which in turn allows cells to form 3D spheroids, resulting in a significant increase in exosome secretion. In one embodiment, the morphology of exosome-producing cells changes to 3D cell spheroids under periodic tensile force. In another embodiment, exosome-producing cells express large amounts of YAP protein under periodic tensile force. Thus, the 3D cell culture method using the periodic tensile bioreactor of this invention can produce exosomes on a large scale.

[0024] The present invention also provides a polynucleotide comprising a sequence encoding a fusion protein, the fusion protein comprising a target protein and an exosome transmembrane protein, the target protein comprising an anti-human leukocyte antigen G (HLA-G) protein.

[0025] Furthermore, the sequence encoding the fusion protein is a nucleic acid sequence. In some embodiments, the sequence encoding the fusion protein includes a first sequence encoding the target protein and a second sequence encoding the exosome transmembrane protein. In one embodiment, the sequence encoding the fusion protein includes SEQ ID NO: 1. In a preferred embodiment, the first sequence containing the anti-HLA-G protein (target protein) includes SEQ ID NO: 1.

[0026] In the present invention, exosome transmembrane proteins are transmembrane proteins or membrane-associated proteins. In another embodiment, exosome transmembrane proteins are CD9, CD53, CD63, CD81, CD82, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, TSPAN14 CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, i Interleukin receptor, immunoglobulin, MHC-I, MHC-II, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD This includes 274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, ARRDC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, or combinations thereof. In preferred embodiments, the exosome transmembrane protein includes CD9, CD63, or CD81. In more preferred embodiments, the exosome transmembrane protein includes CD63.

[0027] In the present invention, the polynucleotide further comprises a promoter which is operably ligated to a sequence encoding a fusion protein. As used herein, “promoter” means a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of a nucleic acid within a cell. The promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. In some embodiments, the promoter can be driven to ligate upstream of the sequence encoding a fusion protein. The promoter that can be ligated is not particularly limited as long as it exhibits promoter activity in the target cell. Examples of promoters that can be ligated to a sequence encoding a fusion protein include, but are not limited to, the EFS promoter, cytomegalovirus (CMV) promoter, CK8 promoter, MHC promoter, MYOD promoter, hTERT promoter, SRalph promoter, SV40 promoter, LTR promoter, CAG promoter, and Roussarcoma virus (RSV) promoter. In one embodiment, the polynucleotide further comprises a promoter used to modulate the sequence encoding a fusion protein.

[0028] Furthermore, the present invention further provides a vector, in which the vector supports polynucleotides. Thus, the polynucleotide having a sequence encoding a fusion protein is present in the vector. In the present invention, one or more vectors are used, each supporting a different sequence fragment.

[0029] In one embodiment, the vector is a plasmid vector, a non-viral vector, or a viral vector. When the vector of the present invention is a plasmid vector, the plasmid vector used is not particularly limited and may be any plasmid vector (such as cloning plasmid vectors and expression plasmid vectors). Plasmid vectors containing the polynucleotide of the present invention are prepared by inserting the polynucleotide of the present invention into a plasmid vector by known methods. In preferred embodiments, viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviruses, or Sendai virus vectors. In this specification, "viral vector" also includes its derivatives. Viral vectors containing the polynucleotide of the present invention can be prepared by known methods. In another embodiment, non-viral vectors include liposomes or lipid nanoparticles.

[0030] In some embodiments, exosome-producing cells are transfected with a polynucleotide-supported vector. Therefore, exosome-producing cells typically contain polynucleotides encoding fusion proteins. Thus, successfully transfected cells can produce single, double, or multiple stable cell lines. Single stable cell lines are advantageous because exosome production is simplified by requiring only the transfection of a single polynucleotide.

[0031] Preferably, exosome-producing cells are stably transfected with a polynucleotide encoding a fusion protein, thereby generating a stable cell line. This advantage ensures consistent production of exosomes of uniform quality and yield. Exosome-producing cells can be genetically modified with at least one polynucleotide using essentially any non-viral or viral method for introducing the polynucleotide into the cell. Preferred methods for introducing polynucleotides into exosome-producing cells include transfection using polycations such as PEI, lipid-based transfection reagents such as lipofectamine (RTM), lentiviral transduction, CRISPR-Cas guide insertion, Flp-In systems, transposon systems, electroporation, DEAE-dextran transfection, and calcium phosphate transfection. The choice of method for introducing polynucleotides into exosome-producing cells depends on various parameters, including the choice of cell source, the nature and characteristics of the vector (e.g., if the vector is a plasmid or minicircle, e.g., linear DNA polynucleotide or mRNA), and the required level of compliance and control. Similarly, immortalization of exosome-producing cells for the creation of stable cell lines can be achieved by using techniques well known in the cell line development field, such as hTERT-mediated immortalization, transcription factor immortalization, E1 / E2 immortalization, or other virus-mediated immortalization techniques. This allows successfully transfected exosome-producing cells to release exosomes containing fusion proteins.

[0032] In an additional aspect, the present invention provides a method for producing exosomes containing a fusion protein, the method comprising: (1) introducing a polynucleotide into an exosome-producing cell, wherein the polynucleotide comprises a sequence encoding a fusion protein, the fusion protein comprises a protein and an exosome transmembrane protein, and the protein comprises an anti-human leukocyte antigen G (HLA-G) protein; and (2) enabling the exosome-producing cell to produce exosomes containing the fusion protein.

[0033] In one embodiment, the exosome-producing cells include mammalian cells. In a preferred embodiment, the exosome-producing cells include embryonic kidney cells. In a more preferred embodiment, the exosome-producing cells include HEK293 cells.

[0034] The present invention also provides a fusion protein comprising a target protein and an exosome transmembrane protein, wherein the target protein comprises an anti-HLA-G protein, and the sequence of the anti-HLA-G protein comprises SEQ ID NO: 2. The peptide sequence of SEQ ID NO: 2 is generated by the nucleic acid sequence of SEQ ID NO: 1.

[0035] The present invention also provides cells comprising a fusion protein, the fusion protein comprising a target protein and an exosome transmembrane protein, the target protein comprising an anti-HLA-G protein, and the sequence of the anti-HLA-G protein comprising SEQ ID NO: 2. In one embodiment, the cells are exosome-producing cells.

[0036] In some embodiments, the target protein directly connects to an exosome transmembrane protein to form a fusion protein. The use of an exosome transmembrane protein allows the target protein to be positioned outside the exosome membrane. This configuration helps to expose the target protein to the outside, enabling binding to molecules on tissue. Suitable exosome transmembrane proteins may be selected from the group including CD63, CD81, CD9, CD82, CD44, CD47, CD55, LAMP2B, ICAM, integrins, ARRDC1, annexins, and any other exosome polypeptides, as well as any combination, derivatives, domains, or regions thereof. A non-limiting example may be a protein such as CD63.

[0037] The present invention also provides an exosome comprising a fusion protein, the fusion protein comprising a target protein and an exosome transmembrane protein, the target protein comprising an anti-HLA-G protein, and the sequence of the anti-HLA-G protein comprising SEQ ID NO: 2. In one embodiment, the exosome transmembrane protein comprises CD9, CD63, or CD81. In a preferred embodiment, the exosome transmembrane protein comprises CD63.

[0038] In one embodiment, the exosome further comprises a therapeutic agent. In a preferred embodiment, the therapeutic agent includes antibodies, antibody fragments, antibody derivatives, single-domain antibodies, intrabodies, single-strand variable fragments, aphid affinity compounds, enzymes, transporters, tumor suppressors, viral or bacterial inhibitors, cellular component proteins, DNA and / or RNA binding proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transporters, nucleotide binding proteins, heat shock proteins, CRISPR-related proteins, and combinations thereof. The therapeutic agent can be taken up by the exosome. Thus, a therapeutic exosome is obtained by introducing / taking up the therapeutic agent into an exosome containing a fusion protein.

[0039] In one embodiment, the therapeutic agent includes an anticancer agent. In a preferred embodiment, the anticancer agent includes a chemotherapeutic agent. In a more preferred embodiment, the anticancer agent includes a microRNA (miR) for treating cancer.

[0040] In another embodiment, the chemotherapeutic agent may be Remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatin, temozolomide, etoposide, cyclophosphamide, temodal, carboplatin, procarbazine, Gliadel, tamoxifen, topotecan, methotrexate, Alisa®, Taxol, Taxotere, fluorouracil, leucovorin, irinotecan, Xeloda, CPT-11, interferon alpha, PEG-induced interferon alpha (e.g., PEG-intron-A), capecitabine, cisplatin This includes latin, Herceptin, Perjeta, epirubicin, pemetrexed, thiotepa, fludarabine, liposomal daunorubicin, cytarabine, paciritaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulfan, prednisone, bortezomib (Velcade®), bisphosphonates, arsenic trioxide, doxorubicin (Doxil®), ganciclovir, adriamycin sodium (Emcyt®), sulindac, etoposide, or combinations thereof. In preferred embodiments, the chemotherapeutic agent includes doxorubicin (DOX), Taxotere (Taxo), cisplatin (Cisp), Herceptin, Perjeta, epirubicin (Epir), cyclophosphamide (Cycl), carboplatin (Carb), gemcitabine (Gemc), pemetrexed (Peme), or a combination thereof. In more preferred embodiments, the chemotherapeutic agent includes doxorubicin.

[0041] In another embodiment, the microRNAs for treating cancer are miR-34a, miR-34b, miR-34c, miR-497, miR-145, miR-206, miR-21, miR-99a, miR-30a, miR-30a, miR-9, miR-210, miR-192, miR-494, miR-221, miR-19a, miR-19b, miR-23b This includes miR-3p, miR-122-5p, miR-193b-3p, miR-141, miR-375, miR-145, miR-196a-5p, miR-200c-3p, miR-1246, miR-1290, miR-21-5p, miR-127-3p, miR-200a, miR-200b, miR-200c, miR-339-5p, or miR-409-3p. In another embodiment, the microRNA for treating cancer includes miR-34a.

[0042] The present invention provides a composition comprising an exosome containing a fusion protein, wherein the fusion protein comprises a target protein and an exosome transmembrane protein, the target protein comprises an anti-HLA-G protein, and the sequence of the anti-HLA-G protein comprises SEQ ID NO: 2.

[0043] The present invention provides a method for treating a subject having cancer, the method comprising administering a composition to a subject having cancer, the composition comprising a therapeutic exosome, the therapeutic exosome comprising a fusion protein and an anticancer agent, the fusion protein comprising a target protein and an exosome transmembrane protein, the target protein comprising an anti-HLA-G protein, the sequence of the anti-HLA-G protein comprising SEQ ID NO: 2. In one embodiment, the exosome transmembrane protein comprises CD9, CD63, or CD81. In a preferred embodiment, the exosome transmembrane protein comprises CD63.

[0044] The present invention provides the use of a composition for preparing a drug for treating cancer, the composition comprising a therapeutic exosome, the therapeutic exosome comprising a fusion protein and an anticancer agent, the fusion protein comprising a target protein and an exosome transmembrane protein, the target protein comprising an anti-HLA-G protein, and the sequence of the anti-HLA-G protein comprising SEQ ID NO: 2. In one embodiment, the exosome transmembrane protein comprises CD9, CD63, or CD81. In a preferred embodiment, the exosome transmembrane protein comprises CD63.

[0045] The term “treat” includes, but is not limited to, reducing, inhibiting or limiting the growth of cancer cells; reducing, inhibiting or limiting the metastasis of cancer cells; reducing, inhibiting or limiting the invasiveness of cancer cells or metastases; and reducing, inhibiting or limiting one or more symptoms of cancer or its metastases. As used herein, “inhibit cancer cell growth” means reducing the rate of proliferation and / or migration of cancer cells, stopping the proliferation and / or migration of cancer cells, killing cancer cells, or reducing cell viability, thereby reducing the rate of cancer cell growth compared to the observed or predicted growth rate of untreated control cancer cells. The term “inhibit growth” may also mean a reduction or disappearance of the size of cancer cells or tumors, as well as a reduction in their metastatic potential. Preferably, such inhibition at the cellular level can reduce size, inhibit growth, and reduce the presence of tumors.

[0046] As used herein, the terms “cancer” or “tumor” refer to or describe a physiological condition in mammals characterized by unregulated cellular growth of a population of cells. Cancer may be a non-solid tumor or a solid tumor. Examples of cancer, but not limited to, include carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include, in particular, breast cancer, oral cancer, medulloblastoma, prostate cancer, squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, ovarian adenocarcinoma, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, gastric cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers, hematological malignancies, acute myeloid leukemia, lymphoma, and leukemia, and melanoma. In one embodiment, cancer includes breast cancer, lung cancer, oral cancer, liver cancer, colorectal cancer, glioblastoma, medulloblastoma, bladder cancer, pancreatic cancer, or ovarian cancer. In a preferred embodiment, cancer includes breast cancer.

[0047] In one embodiment, the subject is an animal, preferably a mammal, and more preferably a human.

[0048] In another embodiment, the anticancer agent comprises a chemotherapeutic agent and / or microRNA (miR) for treating cancer. In a preferred embodiment, the chemotherapeutic agent comprises doxorubicin (DOX), Taxotere (Taxo), cisplatin (Cisp), Herceptin, Perjeta, epirubicin (Epir), cyclophosphamide (Cycl), carboplatin (Carb), gemcitabine (Gemc), or pemetrexed (Peme), or a combination thereof. In a more preferred embodiment, the chemotherapeutic agent comprises doxorubicin. In another embodiment, the microRNA for treating cancer comprises miR-34a.

[0049] In this invention, an anti-HLA-G protein is a protein or peptide having anti-HLA-G function. Therefore, anti-HLA-G proteins can specifically bind to HLA-G molecules. In particular, the sequence of SEQ ID NO: 2 in this invention exhibits a higher affinity for HLA-G. In some embodiments, HLA-G molecules are expressed on cancer cells. Therefore, exosomes containing anti-HLA-G proteins can specifically bind to cancer cells and be used as a means of drug delivery in cancer treatment.

[0050] In some embodiments, therapeutic exosomes are administered with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to any carrier, diluent, or excipient that is compatible with the other components of the formulation and is not harmful to the recipient. A pharmaceutically acceptable carrier can be selected based on the chosen route of administration and standard pharmaceutical practice. The therapeutic exosomes can be formulated into dosage forms according to standard practice in the field of pharmaceutical preparations. Preferred dosage forms, but not limited to, include, for example, liquids, parenteral liquids, injectable liquids, lozenges, suppositories, or suspensions.

[0051] The compositions of the present invention, which contain therapeutic exosomes, can be administered to a target by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, subarachnoid, and intra-articular, or combinations thereof. In preferred embodiments, the composition is administered intravenously or parenterally. In alternative embodiments, the composition is administered by direct injection into a tumor.

[0052] For parenteral administration, the activator can be mixed with a suitable carrier or diluent, for example, but not limited to, water, oil (e.g., vegetable oil), ethanol, physiological saline (e.g., phosphate-buffered saline or physiological saline), aqueous dextrose (glucose) and related sugar solution, glycerol, or glycol such as propylene glycol or polyethylene glycol. Stabilizers, antioxidants, and preservatives may also be added. Suitable antioxidants include, but not limited to, sulfites, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include, but not limited to, benzalkonium chloride, methyl or propylparaben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or non-aqueous solution, dispersion, suspension, or emulsion.

[0053] According to the present invention, the dosage of the composition containing therapeutic exosomes is not particularly limited, as long as it is an effective amount for treatment. It can be appropriately optimized depending on the active ingredient, dosage form, age and weight of the subject, administration schedule, method of administration, etc. The composition is preferably in unit dosage form. In such form, the preparation is divided into unit doses containing an appropriate amount of the active ingredient.

[0054] Methods for treating cancer include administering a therapeutically effective dose of therapeutic exosomes to reduce, inhibit, or limit cancer growth. The term “therapeutically effective dose” refers to an amount sufficient to produce a beneficial or desirable biological and / or clinical outcome. In one embodiment, the “therapeuticly effective dose” is an amount sufficient to inhibit, reduce, or limit the growth of cancer cells compared to the observed or predicted growth rate of an untreated control cancer.

[0055] The dosage of anticancer agents, miRs, or therapeutic exosomes typically ranges from about 0.0001 or 0.001 or 0.01 mg / kg / day to about 1000 mg / kg / day, but can be higher or lower depending on other factors, including the activity of the composition, its bioavailability, the mode of administration, and the various factors discussed above. The dosage and interval can be individually adjusted to provide local and / or systemic concentrations of exosomes sufficient to maintain a therapeutic or prophylactic effect. For example, the composition may be administered once a week, several times a week (e.g., every other day), once a day, or multiple times a day, in particular, depending on the mode of administration, the specific indication being treated, and the judgment of the prescribing physician. Those skilled in the art can optimize the effective local dosage without excessive experimentation. In one embodiment, the therapeutic effective dose of a chemotherapeutic agent is 0.01 to 10 mg / kg body weight. In another embodiment, the therapeutic effective dose of microRNA for treating cancer is 0.01 to 1 mg / kg body weight.

[0056] Furthermore, in the present invention, exosome-producing cells containing fusion proteins can be applied to the periodic tension bioreactor of the present invention. Exosomes are advantageous as delivery vehicles due to various advantages, such as their lack of immunogenicity and their ability to efficiently home into different organs. Therefore, the culture method using the periodic tension bioreactor of the present invention is suitable for the consistent large-scale production of exosomes containing fusion proteins while preserving the biological properties of the exosomes, and these exosomes are suitable as means of delivering drugs (e.g., therapeutic agents). [Examples]

[0057] The present invention can be implemented in many different forms and should not be construed as being limited to the examples described herein. The examples described herein do not limit the scope of the present invention as defined in the claims.

[0058] Materials and methods Vector construction and cell line establishment A sequence encoding an anti-HLA-G VHH / CD63 fusion protein was synthesized and inserted in series into a pcDNA3.4 plasmid (Thermo Scientific) using molecular cloning technology to create an anti-HLA-G-CD63 expression plasmid (pcDNA3.4-αHLA-G-CD63). The DNA sequence of the insert was validated by Sanger sequencing. To establish a stable expression cell line, 293T cells were transfected with pcDNA3.4-αHLA-G-CD63 using lipofectamine 3000 reagent (Thermo Scientific). After transfection, stably transfected 293T cells were selected by culture in G418 (800 μg / mL, Invivogen). 293T cells were able to produce exosomes containing the anti-HLA-G / CD63 fusion protein as cargo exosomes (CARExo).

[0059] The nucleic acid sequence encoding the anti-HLA-G protein is:AGCGCTGGTCACGTGCAGCTGGTGGAAAGCGGCGGCGGCAGCGTGCAAGCCGGCGGCAGCCTGAAGCTGAGCTGCGTGACAAGCGCCTACACCTTCTCCGCTAGCGGCAACTGCATGGGCTGGCTGAGACAAGCCCCCGGCAAGGGCAGAGGGCATCGCCGCCACCTACACAAGAAGCGCCAAGACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCAAGACAACGCCAAGAACACCGTGTACCTGCAGATGAACGGCCTGAAGCCCGAGGACACCGCCACCTACTACTGCGCCGTGGCTAGATGCGCCGGCAGACCCGACAGAAGCACCCTGACAAGCTTCGCCTGGTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGCCTGGAGACCGGT (Sequence ID 1).

[0060] The peptide sequence of the anti-HLA-G protein is:SAGHVQLVESGGGSVQAGGSLKLSCVTSAYTFSASGNCMGWLRQAPGKGREGIAATYTRSAKTYYADSVKGRFTISQDNAKNTVYLQMNGLKPEDTATYYCAVARCAGRPDRSTLTSFAWWGQGTQVTVSSLETG (SEQ ID NO: 2).

[0061] Exo and CARVexo fabrication and characterization To obtain exosomes (Exo or CARExo), the culture medium was centrifuged at 2000 g for 15 minutes to remove cell debris and filtered through 0.22 μm filter paper. The supernatant was then concentrated at 5000 g for 8 minutes by ultrafiltration (Amicon® Ultra, 30 kDa, Merck Millipore, Billerica, MA, USA). The recovered supernatant was sequentially filtered through a 300 kDa membrane using tangential flow filtration (MAP.03 plus TFF system, Lefo Science, Taipei, Taiwan) and resuspended in phosphate buffer (PBS) (pH=7.4) as detailed above. All samples were handled at 4°C for immediate use but stored at -80°C for further use. After isolation, Exo / CARExo were fixed overnight with 1% glutaraldehyde at 4°C. After washing, Exo / CARExo was added to a Holmber carbon-coated grid and negatively stained with phosphotungstic acid aqueous solution for 1 minute. The ultrastructure of Exo / CARExo was analyzed by transmission electron microscopy (TEM, JEOL JEM-1400, Tokyo, Japan).

[0062] These Exo were also assayed by nanoparticle tracking analysis (NTA, ZetaView®, PParticle Metrix GmbH, Meerbusch, Germany) to analyze the size distribution and concentration of exosomes. To analyze biomarkers, Exo / CARExo were immersed in magnetic capture beads coated with anti-CD9 antibody (ab239685, Abcam), anti-CD63 antibody (ab239686, Abcam), and anti-CD81 antibody (ab239687, Abcam) at room temperature for 12 hours and gently mixed. The pre-bound Exo / CARExo were washed and placed in DynaMag® (Invitrogen) for 10 minutes, after which the supernatant was discarded. Bead-conjugated Exo / CARExo were resuspended in buffer and incubated with PE anti-CD9 antibody (E-AB-F1086D, Elabscience, Houston, TX, USA), PE anti-CD63 antibody (ab205540, Abcam), or anti-CD81 antibody (E-AB-F1073D, Elabscience) at 4°C for 2 hours. PE (phycoerythrin)-labeled bead-conjugated Exo was analyzed using BD Accuri® C6 Plus flow cytometry (BD FACS, San Jose, CA, USA). Furthermore, specific Exo / CARExo biomarkers such as CD63, CD81, HSP70, Alix, and β-tubulin (Abcam) were determined using Western blotting.

[0063] Exo uptake assay PKH26-labeled Exo / CARExo in 2 mL of MDA-MB-231 and MCF10A medium (10 9The two cell lines were mixed with Exo ( / mL). First, these two cell lines were grown to 60% confluence in μ-slide wells (ibidi GmbH, Grafelfing, Germany), and then the medium was replaced with medium containing PKH26-labeled Exo. After 6, 12, and 24 hours of incubation, the cells were washed twice with PBS, fixed, and stained with Alexa Fluor® 488 phalloidin and DAPI. After washing, images of the cells were taken using a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

[0064] Cell viability Cells were cultured in 96 wells containing Exo / CARExo for harvesting at different time points. The culture medium was collected and washed with PBS solution to remove any residual medium. Tetrazolium dye MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was mixed with fresh medium in a 1:9 ratio to assess cell viability by acting on the respiratory chain. After a 3-hour reaction, the MTT agent was removed, DMSO was added and allowed to stand for 20 minutes to dissolve the formazan crystals, which were then redistributed evenly into the 96 wells. Absorbance was quantified by measuring at a wavelength of 570 nm to assess cell viability.

[0065] Immunofluorescence MDA-MB-231, MCF-7 and MCF-10A cells (density 5x10 4Cells (1 / mL) were seeded in 35mm dishes with coverslips and allowed to adhere for 12 hours. MDA-MB-231, MCF-7, and MCF-10A cells were cultured at 37°C for 6 hours in the presence of one of the nanoprobes (0 μM and 5 μM). The cells were washed once in PBS, fixed, and simultaneously permeabilized with 4% paraformaldehyde in PBS containing 1% Triton X-100. Next, the cells were quenched with PBS containing 0.1 M glycine and blocked overnight at 4°C with 3% (wt / vol) BSA. The fixed and permeabilized cells were stained with primary and secondary antibodies as instructed. Then, the cells were stained with DAPI solution at room temperature for 5 minutes. Goat anti-mouse IgG h&L (Alexa Fluor® 647) (ab150115) and goat anti-rabbit IgG h&L (Alexa Fluor® 488) (ab150077) were obtained from Abcam (Cambridge, UK). Cell morphology was observed using a laser confocal microscope (Zessis, LSM800, Germany).

[0066] Preparation of Exo / CARExo containing miRNA and doxorubicin (DOX) To incorporate exogenous cargo into Exo / CARExo, miR-34a and doxorubicin were transfected by electroporation. The amount of Exo / CARExo was 1 x 10⁻¹⁶. 9(Measured by NTA), and mixed with a buffer containing miR-34a before electroporation. After electroporation, Exo / CARExo was left standing at 37 °C for 1 hour. Then, it was mixed with 1 mg of doxorubicin, reacted at 37 °C for 60 minutes, then switched to 4 °C and reacted for 30 minutes. Then, 20 μL of ExoQuick-TC (System Bioscience) was added and mixed well, and reacted at 4 °C for 16 hours. After the reaction, centrifuged at 1500 x g at 4 °C for 30 minutes, the supernatant was aspirated and removed, 200 μL of PBS was added for washing and removed by centrifugation, the above PBS washing was repeated twice, and finally 100 μL of PBS was added to uniformly disperse the exosomes. When completed, the number of Exo / CARExo was re-analyzed by NTA, and the number of Exo / CARExo lost during drug delivery was analyzed.

[0067] Biodistribution The present invention evaluated the tumor targeting effect of Exo / CARExo in vivo using a tumor xenograft mouse model (NOD / SCID gamma mice). PBS, Exo, and CARExo were injected via the tail vein at 100 μL (5 x 10^10 Exo / CARExo). The mice were scanned at different time points and sacrificed at 24 hours.

[0068] In vivo antitumor effect The xenograft tumor model was established by injecting 0.1 mL of MDA-MB-231 cell suspension (5 x 10 6 / 100 μL) into the right flank NOD / SCID gamma (NSG) nude mice. Established MDA-MB-231 tumor (150 - 200 mm 3 ) bearing mice were randomly classified into groups and the groups were treated as follows: (i) PBS as a control; (ii) Exo-miR; (iii) Exo-drug; (iv) Exo-drug-miR (drug equivalent was 5 mg / kg, miR equivalent was 0.1 nmoL / kg). The drug was injected weekly via the tail vein for 7 consecutive weeks. The body weight of the mice was measured, and the tumors were measured with calipers every 4 days. The tumor volume was calculated using the following formula: tumor volume = (length x width x width) / 2.

[0069] Tissue recovery and hematoxylin-eosin (H&E) staining Mice were anesthetized by intraperitoneal injection of chloral hydrate aqueous solution (3% w / v) at a dose of 1 mL / kg. They were then sacrificed, blood samples were taken, and the abdominal cavity was exposed. Liver, lung, spleen, heart, and kidney samples were collected and temporarily stored in 10% neutral buffered formalin (NBF) before hematoxylin-eosin staining.

[0070] NBF (pH 7.2-7.4) was prepared from 100 mL of a 40% v / v aqueous solution of formaldehyde, 6.5 g of anhydrous disodium hydrogen phosphate, 4.0 g of sodium dihydrogen phosphate, and 900 mL of distilled water. Tissues from the liver, spleen, lungs, heart, and kidneys were collected from a 10% NBF solution. Tissue sections were dehydrated, embedded in paraffin, cut, and stained with hematoxylin and eosin.

[0071] Building a 3D Spheroid Model All cells were cultured in their respective appropriate media. The culture media were changed every 2 or 3 days. Prior to decellularization of the lung ECM-embedded culture, each cell type was recovered by treatment with EDTA (Gibco) solution containing 0.25% trypsin, and the cells were recovered by centrifugation at 1200 rpm for 10 minutes, and the cells were dispersed in each culture medium. The cell suspensions were then prepared for each cell type at a final concentration of 5 x 10⁶. 6 cells ml -1 The cells were mixed with ECM pregel solution (4 wt%) to achieve the desired consistency. Aliquots of each cell-mounted pregel solution were transferred to the wells of a multi-well plate and incubated in a 37°C incubator for 1 hour. After gelation, culture medium for cancer or endothelial cells was added to the wells. Each culture medium was replaced every 2 or 3 days.

[0072] Preparation of FGelMa hydrogel containing cells with growth-stimulating structures In this invention, a hydrogel with a growth-stimulating structure was fabricated via a mold-forming process. Considering that the cell-containing hydrogel with the growth-stimulating structure should be compatible with a tensile bioreactor, three components—Pluronic F-127 solution, GelMa, and cell-containing FGelMa—were used to manufacture the construct. The indentation mold was designed and printed using a BioX bioprinter (Cellink, Sweden) with Pluronic F-127 (Sigma-Aldrich) solution at a concentration of 30% by weight in distilled water. Figures 16A-16C show representative unit cells of the growth-stimulating construct. For mold printing, a stereolithography (STL) model was designed and developed using SolidWorks. The STL file was converted to a g-code file and used sli3er software to determine the bioprinter's movement and printing path. After filling a syringe barrel equipped with a 150 μm inner diameter Luer lock nozzle with fugitive ink, the fugitive ink was deposited onto a glass slide using a syringe with a transfer speed of 25 mm / s and an air pressure of 170 kPa to form a filament with a diameter of approximately 250 μm. The fugitive molds of the growth-stimulating constructs were fabricated layer by layer (150 μm per layer) until the final thickness of the construct reached 2.1 mm. Next, heated (37°C) PBS containing 15 wt% GelMa and 0.5 wt% I-2959 was placed in the molds to form a fixed section with two openings on each side, which could be connected to a tensile bioreactor (ATMS Boxer QQA Cyclic Stretch Culture System, Genemessenger, Kaohsiung, Taiwan) (Figure 16A). Cell-containing FGelMa was obtained by mixing HEK293T (or other normal cell lines and primary cells) with PBS containing 0.5% I-2959 and 10% by weight of FGelMa. Confluent cells cultured in culture medium were trypsinized, centrifuged, and measured to a density of 5x10⁶. 6The cells were resuspended in a warmed FGelMa solution at a concentration of cells / mL. The cell-containing hydrogel was then incorporated into the growth-stimulating portion of the sample. The sample was incubated at 37°C for 1 minute to liquefy the GelMa and FGelMa. After exposure to UV irradiation (365 nm) for 90 seconds, the sample was immersed in cold (4°C) PBS for 10 minutes to dissolve the chromogenic F-127 (Figure 16B). To apply periodic tensile force, the sample was immobilized in a tensile bioreactor and 8 mL of culture medium was added. During the culture period, a 20% tensile strain was continuously applied to the cell-containing hydrogel at a frequency of 0.48 Hz. Samples cultured under static conditions served as the control group (Figure 16C). All in vitro experiments were performed in a cell culture incubator at 37°C with 5% CO2 and 100% humidity, and the culture medium was replaced every two days.

[0073] The synthesis and characterization of FGelMa, as well as the design and application of a tensile bioreactor, are described in Yi-Wen Chen et al. (Materials & Design, Volume 195, October 2020, 108982), which are incorporated herein by reference in their entirety.

[0074] YAP inhibitors and YAP treatment In previous results, the present invention suggested that the presence of tensile stimulation of HEK293T is mediated through the cellular activation of YAP protein, a mechanosensitive transcription activator that plays a crucial role in cell behavior. Therefore, the present invention ultimately exposed HEK293T-containing growth-stimulating scaffolds to verteporfin (MedChemExpress, Monmouth Junction, NJ, USA), a YAP inhibitor. After specific days of culture, the present invention evaluated the role of YAP in tensile stimulation using immunofluorescence staining and bone formation-related proteins.

[0075] result The characteristics of exosomes were examined, both those treated with cargo engineering and those without. Morphological results were confirmed by TEM imaging of Exo and CARExo isolated using tangential flow filtration (TFF) technology. The results showed similar levels of exosome size even with cargo engineering (Figure 1A). They clearly exhibited vesicular hollowness and surface membrane characteristics. NTA analysis showed separate main diameter peaks at 110 nm (Exo) and 120 nm (CARExo) (Figure 1B). These results indicate that the morphology of exosomes remains stable even after treatment with cargo engineering technology.

[0076] The presence of CD9, CD63, CD81, HSP71, and Alix, which are generally enriched on the exosome surface membrane, was analyzed using Western blotting analysis with anti-CD9, anti-CD63, anti-CD81, anti-HSP70, and anti-Alix (Figure 2A). The cytoplasmic marker β-tubulin was not present in the exosomes. The results of this invention demonstrate that CARexo expressed similar exosome characteristic markers. Flow cytometry analysis showed that the levels of exosome markers CD9, CD63, and CD81 were detected in the Exo group (Figure 2B). Furthermore, surface modification of exosomes by cargo engineering techniques did not affect the performance of the parent exosomes.

[0077] Numerous studies have shown that exosomes can be used as ideal cargo for therapeutic effects against specific cellular targets (Figure 3). Therefore, this invention further investigated the uptake of CARexo in the nucleus of MDA-MB-231 cells. CARexo was stained with the exosome tracker reagent PKH67 (green) and then co-cultured with MDA-MB-231 cells for 24 hours. Subsequently, the cells were further stained with the F-actin cytoskeleton reagent (red) and the nuclear reagent DAPI (blue). These images, as observed by confocal microscopy, showed that exosomes were incorporated into the nuclear layer (YZ sections). Therefore, the high potential of CARexo for precise cancer treatment was a key factor.

[0078] Furthermore, to further confirm that CARexo can specifically target breast cancer (MDA-MB-231), the present invention also used normal mammary cells (MCF10A) for comparison (Figure 4). MDA-MB-231 and MCF-10A were incubated separately with Exo and CARexo at different time points, and immunofluorescence detection was performed at 6 hours, 12 hours, and 24 hours. The results showed that 231 cells had good affinity for CARexo. At 12 hours, there was potential for large amounts of CARexo uptake, but the amount of Exo was relatively small.

[0079] Exosome targeting rates were measured by flow cytometry. MDA-MB-231 and MCF-10A were treated with CARExo and Exo for 6 hours and 24 hours, respectively (Figure 5). At 6 hours, there was no difference in expression between the two groups. However, treatment with CARExo for 24 hours resulted in a significant increase in the MDA-MB-231 subpopulation in this invention, while Exo showed almost no change. CARExo accurately targeted MDA-MB-231 and was more effective than MCF-10A.

[0080] In experiments where CARExo was co-cultured with cancer cells (MDA-MB-231, red) and normal cells (HDF, light green), it was found that CARExo (green) was concentrated in the cancer cells, further confirming that CARExo has specificity for cancer cells (Figure 6).

[0081] Figure 7 shows the evaluation of cytotoxicity of CARExo, Exo, and control (Ctl) against MD-MBA-231, MCF10A cells. Neither CARExo nor Exo showed any activity at 6, 12, 24, and 48 hours of co-culture, indicating that these exosomes were completely cytotoxic. These results were representative of three independent experiments.

[0082] To further evaluate the in vivo distribution of CARExo in relation to its in vivo therapeutic potential, this invention constructed a xenograft mouse model using MDA-MB-231 cells (Figure 8). Consistent with in vitro results, CARExo significantly targeted tumors, and therefore little signal was detected in the lungs and spleen, which are storage sites for normal exosomes.

[0083] Furthermore, the ideal electroporation parameters were in the range of 600mV to 800mV, which allowed exosomes to stably deliver miR-34a (Figure 9). As a result, 700mV showed the best delivery efficiency, and CARexo was able to detect 30ng of miR-34a. This comprehensive efficiency suggests a high potential for drug delivery applications. CARexo delivered the cancer-targeting drug doxorubicin (Dox) with high efficiency at 700mV. The results further showed excellent efficiency when Dox was delivered together with 20ng of miR-34a. These results demonstrate that these electroporation parameters allow exosomes to stably deliver two drugs simultaneously.

[0084] Next, in this invention, the test group was further increased to six groups: control, drug, miR, CARExo, CARExo-drug, and CARExo-miR-drug, and the results of verifying cancer cell killing efficacy were identified (Figure 10). After 24 and 48 hours, the Exo-miR-drug showed the best cell killing effect compared to the other groups. On the other hand, the effect of CARExo or the drug alone was approximately 50%. It can be confirmed that the exosome cocktail can effectively and rapidly kill cancer cells on the first attempt.

[0085] Next, the present invention was stained with CARESO and miR-34a trackers, respectively (Figure 11). As expected, we verify here that CARESO (green) encapsulated miR-34a (red), which may exhibit colocalization.

[0086] This invention further demonstrates that CARexo exosomes have an additive effect on tumor suppression in vivo (Figures 12A-12B). Four groups were designed: control (Ctl), CARexo-miR (cargo exosome containing miR-34a), CARexo-drug (cargo exosome containing doxorubicin), and CARexo-drug-miR (cargo exosome containing miR-134a and doxorubicin). Animal studies involved subcutaneous injection of CARexo-drug-miR into mice and observation at different time points. The inhibitory effect on tumor formation was found to be superior to the other groups (Figure 12A). Tumor volume and weight were significantly reduced in the CARexo-drug-miR group. Next, this invention aimed to confirm cytotoxicity in other organs. Therapeutic studies focused on the liver and lungs, as well as other organs such as the heart, kidneys, and spleen. HE staining showed that the organs described above retained their original morphology (Figure 12B). CARExo-drug-miR proved to be a safe and precise target at the tumor site.

[0087] Figure 13 shows that the present invention also tested the efficacy of CARexo using different cancer models: MB11 (glioblastoma), A549 (non-small cell lung cancer), HepG2 (hepatocellular carcinoma), OECM (oral squamous cell carcinoma), SKOV3 (ovarian cancer cell line), and T24 (bladder cancer).

[0088] In addition, the present invention designed seven groups: control (Ctl), DOX (doxorubicin), LipoDox (doxorubicin-containing liposome), Exo (exosome), CARExo (cargo exosome), DOX@Exo (doxorubicin-containing exosome), and DOX@CARExo (doxorubicin-containing cargo exosome), and tested different cancer cell lines. Similar results were observed with different cancer cell lines (Figure 14). In addition to breast cancer (MB231), lung cancer (HCC827), oral cancer (OECM), liver cancer (HepG2), colorectal cancer (CaCO2), pancreatic cancer (MIAPaCa-2), medulloblastoma (U87), bladder cancer (T24), and ovarian adenocarcinoma (SKOV3), among others. DOX@CARExo was found to exhibit good cytotoxicity. Furthermore, in normal cells, the cytotoxicity of CARExo was lower than that of DOX and Lipo-DOX.

[0089] Furthermore, the present invention can also function as a carrier for transporting different chemotherapeutic agents (e.g., doxorubicin (DOX), taxotere (Taxo), cisplatin (Cisp), epirubicin (Epir), cyclophosphamide (Cycl), carboplatin (Carb), gemcitabine (Gemc), and pemetrexed (Peme)) via exosomes to kill cancer cells (Figure 15). As a result, chemotherapeutic agents contained in CARexo showed a higher killing effect on cancer cells (MDA-MB-231) than EXO, exhibiting the same trend as previous results with DOX.

[0090] Hydrogel scaffolds with different concentrations (10%, 12.5%, and 15%) were subjected to periodic tensile stress at various time points, resulting in significant changes in exosome secretion efficiency (Figure 17). The 15% FGelMa scaffold released more exosomes than the 12.5% ​​and 10% scaffolds. Furthermore, the present invention also evaluated the efficiency of exosome secretion per cell. After one month of periodic tensile stress treatment, the amount of exosome secreted per cell was approximately 1.03 x 10⁶ in the 15%, 11.5%, and 10% FGelMa hydrogel scaffolds, respectively. 6 , 0.92 x 10 per cell 6, and 0.81 x 10 per cell 6 The present invention also compared this experiment with a market bioreactor specifically designed for exosome production. These results show that the FiberCell system can continuously harvest exosomes for extended periods, but the yield after one month is approximately 0.44 x 10¹⁶ cells. 6 The cells were found to be far fewer in number than in the system of the present invention. The other system, CelCradle®, was a type of bioreactor similar to suspension culture. Because exosomes could not be recovered for a long period, the test could only be conducted for about two weeks, and the yield was also lower than in the system of the present invention.

[0091] The present invention further identified changes in cell inclusion on a hydrogel scaffold induced by stretching stimulation using fluorescent staining of the cytoskeleton (F-actin, green), a mechanosensing marker (YAP, red), and the nucleus (blue) (Figure 18). As shown in Figure 18, after stretching cells by periodic tension for three days, the cells transformed from single cells to self-organizing three-dimensional cell spheroids, while the cells in the static culture group remained single cells. Furthermore, the three-dimensional cell spheroids formed by mechanical force stimulation expressed a large amount of YAP protein around the spheroid, while static culture cells hardly expressed it. Therefore, the present invention hypothesizes that cells stimulated by mechanical force expressed a large amount of YAP protein, enhancing exosome secretion after the formation of three-dimensional spheroids.

[0092] Next, a YAP protein (verteporfin) inhibitor was added via a periodic tensile stimulation process. Fluorescence staining showed that in the group without the inhibitor, YAP protein expression, cell spheroid morphology, and the amount of secreted exosomes were consistent with previous results (Figure 19A). The cell morphology of the group with the inhibitor was that of single cells unable to self-organize and form three-dimensional spheroids, similar to statically cultured cells, and exosome secretion was also significantly reduced to 5% of that of the periodic tensile stimulation group (Figure 19B). Therefore, it was hypothesized that under three-dimensional culture conditions, continuous mechanical stimulation can activate the YAP protein, causing cells to form three-dimensional spheres and significantly increasing exosome secretion.

[0093] To confirm the appearance of exosomes secreted by cells after static / dynamic stretching, the present invention isolated extracellular vesicles using a standard TFF protocol (Figure 20). Transmission electron microscopy and Nanosight size distribution analysis revealed a population of small, round particles ranging from 50 to 350 nm, with a main peak at 122 nm. Western blot analysis showed significant enrichment of tetraspanin exosome markers CD9, CD63, CD81, TSG101, and the cytoplasmic marker β-tubulin under dynamic stretching. More moderate enrichment of other exosome markers, CD9, CD63, CD81, and TSG101, was observed under static stretching. These results, taken together, confirmed the successful isolation of exosome-like particles from dynamic stretching.

[0094] Those skilled in the art will understand the above concepts as a description of the methods used to transmit deposited application information. Those skilled in the art will recognize that these are merely examples and that many equivalents are possible.

Claims

1. A method for promoting exosome production, the method comprising: (a) providing a periodic tension bioreactor, the periodic tension bioreactor comprising a culture chamber, a biocompatible polymer material having a growth-stimulating structure, and a plurality of tension components, wherein the biocompatible polymer material and the plurality of tension components are arranged in the culture chamber, the biocompatible polymer material contains exosome-producing cells, the exosome-producing cells are incorporated into the biocompatible polymer material, both ends of the biocompatible polymer material are connected by the plurality of tension components, and the exosome-producing cells are human embryonic kidney cells, mesenchymal stem cells, human umbilical vein endothelial cells, M2 polarized macrophages, fibroblasts, or chondrocytes; (b) repeatedly stretching the biocompatible polymer material with the plurality of tension components for applying periodic tension; and (c) recovering the exosomes released from the exosome-producing cells by the periodic tension applied by the plurality of tension components.

2. The method according to claim 1, wherein the biocompatible polymer material includes a methacryloyl polymer.

3. The method according to claim 1, wherein the concentration of the biocompatible polymer material is in the range of 0.1 to 50% by weight.

4. The method according to claim 1, wherein the biocompatible polymer material includes a plurality of pores.

5. The method according to claim 1, wherein the periodic tensile force applied by the plurality of tensile components is a tensile strain of 2.5 to 50%.

6. The method according to claim 1, wherein the periodic tensile force applied by the plurality of tensile components is in the frequency range of 0.1 to 4 Hz.

7. The method according to claim 1, wherein the recovery of the exosomes in step (c) is performed one to two months after the application of the periodic tensile force.

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