Umbilical cord mesenchymal stem cell exosome targeting delivery system based on adenovirus-mediated EpCAM modification and application of umbilical cord mesenchymal stem cell exosome targeting delivery system

Abstract

The invention discloses an umbilical cord mesenchymal stem cell exosome targeting delivery system based on adenovirus-mediated EpCAM modification and application thereof, the system introduces an anti-EpCAM single-chain antibody gene into umbilical cord mesenchymal stem cells through an adenovirus vector, so that an anti-EpCAM single-chain antibody is stably expressed on the surface of exosome secreted by the umbilical cord mesenchymal stem cells; accurate targeting delivery of epithelial tissues is realized by using antigen-antibody specific binding, the problem of insufficient targeting of natural exosomes is effectively solved, and the exosome has remarkable advantages in epithelial tissue disease treatment and has good application prospects and important transformation significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and specifically relates to an adenovirus-mediated EpCAM-modified umbilical cord mesenchymal stem cell exosome targeted delivery system and application thereof. BACKGROUND

[0002] Exosomes derived from mesenchymal stem cells (MSCs) have good biocompatibility and biological activity, and have shown broad application prospects in various disease treatment fields, including lung injury repair, immune regulation, bone and joint disease treatment, and tumor treatment, etc. They can play a role by regulating related protein expression, balancing immune cell subpopulations, regulating signaling pathways, or serving as drug carriers. To improve the treatment accuracy of exosomes, existing targeting modification technologies mainly include genetic engineering methods (transfecting exosome-derived cells to express targeting peptides or antibody fragments), chemical coupling methods (such as using high molecular weight modification of targeting peptides), and membrane fusion technology (fusing exosomes with modified targeting molecules with liposomes). Meanwhile, epithelial cell adhesion molecule (EpCAM) has been applied in the detection of circulating tumor cells and targeted therapy of tumors (such as antibody conjugated drugs and CAR-T cell therapy), and adenovirus vectors have been used for gene therapy and targeted delivery research due to their good gene delivery effect.

[0003] However, the existing technology still has many deficiencies. Natural exosomes lack tissue specificity, and the enrichment efficiency of the target site is low. The existing targeting peptides have limited affinity for epithelial tissue and are difficult to penetrate the epithelial barrier of the lung, intestine, etc. Chemical coupling methods can easily affect the integrity of exosomes and have large batch differences. Membrane fusion technology is complex and difficult to produce on a large scale. Traditional genetic engineering methods have low transfection efficiency, resulting in unstable expression. In addition, the tight junctions and mucus barriers of the lung and intestinal epithelium make it difficult for traditional nanocarriers to effectively penetrate, and there is a lack of targeting molecules with high affinity and specificity for epithelial tissue. Some exogenous targeting molecules can trigger an immune response, and chemical modification can introduce toxic substances. At the same time, the application of EpCAM is currently limited to tumor diagnosis and treatment, and its potential in epithelial tissue targeted delivery has not been developed. Moreover, existing EpCAM antibody modification methods are complex and costly, making them difficult to apply to exosome modification. SUMMARY

[0004] In view of this, in order to overcome the above technical problems existing in the prior art, the purpose of the present application is to provide an adenovirus-mediated EpCAM-modified umbilical cord mesenchymal stem cell exosome targeted delivery system and application thereof.

[0005] The technical scheme adopted by the present application to achieve the above-mentioned application purposes is as follows:

[0006] The first aspect of the present application provides an adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosome.

[0007] Further, the umbilical cord mesenchymal stem cell exosome stably expresses an anti-EpCAM single-chain antibody on the surface.

[0008] Further, the amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO: 1.

[0009] Optionally, the adenovirus is an Ad5 type adenovirus.

[0010] Optionally, the umbilical cord mesenchymal stem cell is a human umbilical cord mesenchymal stem cell, a mouse umbilical cord mesenchymal stem cell, a rat umbilical cord mesenchymal stem cell, a rabbit umbilical cord mesenchymal stem cell, a pig umbilical cord mesenchymal stem cell, or a dog umbilical cord mesenchymal stem cell.

[0011] Optionally, the umbilical cord mesenchymal stem cell exosome expresses CD9, CD63, CD81, HSP70, and / or TSG101 proteins.

[0012] Optionally, the particle size of the umbilical cord mesenchymal stem cell exosome is 70-150 nm.

[0013] In the present application, the epithelial cell adhesion molecule (EpCAM, same as Epcam) is a transmembrane glycoprotein widely expressed on the surface of normal epithelial cells, which plays an important role in physiological processes such as cell adhesion, proliferation, and differentiation. Abnormal high expression of EpCAM is often found in various diseases of epithelial tissue origin, especially in epithelial tumors such as lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, etc., which is closely related to tumor cell proliferation, invasion, metastasis, and prognosis. Due to the specific expression characteristics of EpCAM in diseased epithelial tissues, it is not only an important biomarker for clinical diagnosis and prognosis evaluation, but also a key target for targeted therapy, providing a molecular basis for precise treatment of epithelial tissue diseases.

[0014] In a specific embodiment of the present application, the umbilical cord mesenchymal stem cell is a human umbilical cord mesenchymal stem cell.

[0015] The second aspect of the present application provides a method for preparing an adenovirus-mediated anti-EpCAM single-chain antibody modified umbilical cord mesenchymal stem cell exosome.

[0016] Further, the method comprises the following steps:

[0017] (1) Constructing an adenovirus vector: constructing an adenovirus vector carrying an anti-EpCAM single-chain antibody;

[0018] (2) Packaging and purification: obtaining an adenovirus carrying an anti-EpCAM single-chain antibody by packaging and purification;

[0019] (3) Infection and harvesting: umbilical cord mesenchymal stem cells are infected, and the supernatant of the infected cells is collected, and the umbilical cord mesenchymal stem cell exosomes are isolated and harvested.

[0020] Further, the amino acid sequence of the anti-EpCAM single-chain antibody in step (1) is shown in SEQ ID NO: 1;

[0021] Optionally, the adenovirus vector is an Ad5 type adenovirus vector;

[0022] Optionally, the Ad5 type adenovirus vector is an Ad5 type adenovirus vector with deleted E1 / E3 region;

[0023] Optionally, the nucleotide sequence of the Ad5 type adenovirus vector carrying the anti-EpCAM single-chain antibody is shown in SEQ ID NO: 7.

[0024] Further, step (2) comprises the following steps: the adenovirus vector carrying the anti-EpCAM single-chain antibody is packaged in a host cell, and purified by centrifugation;

[0025] Optionally, the host cell is a HEK293 cell, a HEK293T cell, a HEK293F cell, a HEK293E cell, a HEK293-6E cell, a HEK293FT cell, a HEK293H cell, a HEK293S cell, or a PER.C6 cell;

[0026] Optionally, the centrifugation method is CsCl density gradient centrifugation, iodixanol density gradient centrifugation, or sucrose density gradient centrifugation;

[0027] Optionally, the adenovirus in step (2) is an anti-EpCAM single-chain antibody-carrying adenovirus obtained by packaging and purifying the adenovirus vector carrying the anti-EpCAM single-chain antibody.

[0028] Further, step (3) comprises the following steps: umbilical cord mesenchymal stem cells are infected with the adenovirus carrying the anti-EpCAM single-chain antibody, the supernatant of the infected cells is collected, and the umbilical cord mesenchymal stem cell exosomes are isolated and harvested;

[0029] Optionally, the multiplicity of infection MOI of the infection is 50-150;

[0030] Optionally, the multiplicity of infection MOI of the infection is 100;

[0031] Optionally, the supernatant of the infected cells is collected 48-96 hours after infection;

[0032] Optionally, the supernatant of the infected cells is collected 72 hours after infection;

[0033] Optionally, the umbilical cord mesenchymal stem cell exosome is separated by centrifugation.

[0034] Optionally, the centrifugation is ultracentrifugation.

[0035] Optionally, the ultracentrifugation is performed under the following conditions: (100-500) × g centrifugation for 5-15 min to remove dead cells and cell debris; (1500-3000) × g centrifugation for 5-15 min to remove microvesicles and large-diameter vesicles; 100000-150000 × g ultracentrifugation for 60-120 min to precipitate exosomes.

[0036] Optionally, the ultracentrifugation is performed under the following conditions: 300 × g centrifugation for 10 min to remove dead cells and cell debris; 2000 × g centrifugation for 10 min to remove microvesicles and large-diameter vesicles; 120000 × g ultracentrifugation for 90 min to precipitate exosomes.

[0037] Optionally, the step (3) further comprises the following step: after resuspension in PBS, filtering to remove bacteria, to obtain the umbilical cord mesenchymal stem cell exosome stably expressing the anti-EpCAM single-chain antibody on the surface.

[0038] Optionally, the umbilical cord mesenchymal stem cell in step (3) is human umbilical cord mesenchymal stem cell, mouse umbilical cord mesenchymal stem cell, rat umbilical cord mesenchymal stem cell, rabbit umbilical cord mesenchymal stem cell, pig umbilical cord mesenchymal stem cell or dog umbilical cord mesenchymal stem cell.

[0039] In a specific embodiment of the present application, the umbilical cord mesenchymal stem cell is human umbilical cord mesenchymal stem cell.

[0040] In a specific embodiment of the present application, the human umbilical cord mesenchymal stem cell is obtained by the following method: (1) enzyme digestion: the umbilical cord tissue is cut into 1-2 mm 3 size, and is mixedly digested with 0.1% collagenase II and 0.1% hyaluronidase for 30-45 min, and a single cell suspension is obtained by filtering through a 70 μm cell screen; (2) culture expansion: serum-free medium is used, and the culture is carried out at 37℃, 5% CO2, the medium is changed every 3 days, and when the confluence reaches 80%, the cells are passaged by trypsinization; (3) identification: the differentiation abilities of osteogenesis, chondrogenesis and adipogenesis in vitro are verified.

[0041] The third aspect of the present application provides a method for preparing an adenovirus-mediated anti-EpCAM single-chain antibody modified umbilical cord mesenchymal stem cell.

[0042] Further, the method comprises the following steps:

[0043] (1) constructing an adenovirus vector: constructing an adenovirus vector carrying an anti-EpCAM single-chain antibody;

[0044] (2) packaging and purification: obtaining an adenovirus carrying an anti-EpCAM single-chain antibody through packaging and purification;

[0045] (3) infection: infecting umbilical cord mesenchymal stem cells to obtain an adenovirus-mediated anti-EpCAM single-chain antibody modified umbilical cord mesenchymal stem cell;

[0046] Optionally, the amino acid sequence of the anti-EpCAM single-chain antibody in step (1) is shown as SEQ ID NO: 1;

[0047] Optionally, the adenovirus vector is an Ad5 type adenovirus vector;

[0048] Optionally, the Ad5 type adenovirus vector is an Ad5 type adenovirus vector with deleted E1 / E3 region;

[0049] Optionally, the nucleotide sequence of the Ad5 type adenovirus vector carrying an anti-EpCAM single-chain antibody is shown as SEQ ID NO: 7;

[0050] Optionally, step (2) comprises the following steps: packaging the adenovirus vector carrying an anti-EpCAM single-chain antibody in a host cell, and purifying by centrifugation;

[0051] Optionally, the host cell is a HEK293 cell, a HEK293T cell, a HEK293F cell, a HEK293E cell, a HEK293-6E cell, a HEK293FT cell, a HEK293H cell, a HEK293S cell, or a PER.C6 cell;

[0052] Optionally, the centrifugation method is a CsCl density gradient centrifugation method, an iodixanol density gradient centrifugation method, or a sucrose density gradient centrifugation method;

[0053] Optionally, the adenovirus in step (2) is an adenovirus carrying an anti-EpCAM single-chain antibody obtained through packaging and purification of the adenovirus vector carrying an anti-EpCAM single-chain antibody;

[0054] Optionally, step (3) comprises the following steps: infecting umbilical cord mesenchymal stem cells with the adenovirus carrying an anti-EpCAM single-chain antibody to obtain an adenovirus-mediated anti-EpCAM single-chain antibody modified umbilical cord mesenchymal stem cell;

[0055] Optionally, the multiplicity of infection MOI of the infection is 50-150;

[0056] Optionally, the multiplicity of infection MOI of the infection is 100;

[0057] Optionally, the umbilical cord mesenchymal stem cells mentioned in step (3) are human umbilical cord mesenchymal stem cells, mouse umbilical cord mesenchymal stem cells, rat umbilical cord mesenchymal stem cells, rabbit umbilical cord mesenchymal stem cells, porcine umbilical cord mesenchymal stem cells, or canine umbilical cord mesenchymal stem cells.

[0058] The fourth aspect of the present invention provides any of the following products:

[0059] (1) An adenovirus vector carrying an anti-EpCAM single-chain antibody, wherein the amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO:1;

[0060] Optionally, the adenovirus vector is an Ad5 type adenovirus vector;

[0061] Optionally, the Ad5 adenovirus vector is an Ad5 adenovirus vector with the E1 / E3 region deleted;

[0062] Optionally, the nucleotide sequence of the Ad5 adenovirus vector carrying the anti-EpCAM single-chain antibody is shown in SEQ ID NO:7;

[0063] (2) An adenovirus carrying an anti-EpCAM single-chain antibody, wherein the adenovirus is an adenovirus carrying an anti-EpCAM single-chain antibody obtained by packaging and purifying the adenovirus vector carrying the anti-EpCAM single-chain antibody described in (1).

[0064] (3) An adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosome prepared by the method described in the second aspect of the present invention;

[0065] Optionally, the umbilical cord mesenchymal stem cell exosomes stably express anti-EpCAM single-chain antibodies;

[0066] Optionally, the umbilical cord mesenchymal stem cell exosomes express CD9, CD63, CD81, HSP70 and / or TSG101 proteins;

[0067] Optionally, the particle size of the umbilical cord mesenchymal stem cell exosomes is 70-150 nm;

[0068] (4) An adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell prepared by the method described in the third aspect of the present invention;

[0069] (5) A targeted delivery system for the treatment of epithelial tissue diseases, the targeted delivery system comprising umbilical cord mesenchymal stem cell exosomes modified with an adenovirus-mediated anti-EpCAM single-chain antibody as described in (3);

[0070] Optionally, the targeted delivery system further includes a therapeutic drug carried in or on an exosome;

[0071] Optionally, the therapeutic drug is one or more of small molecule drugs, nucleic acid drugs, and protein drugs used to treat epithelial tissue diseases;

[0072] (6) A pharmaceutical composition for treating epithelial tissue diseases, the pharmaceutical composition comprising umbilical cord mesenchymal stem cell exosomes modified with an adenovirus-mediated anti-EpCAM single-chain antibody as described in (3);

[0073] Optionally, the pharmaceutical composition further comprises a therapeutic agent for treating epithelial tissue diseases;

[0074] (7) A pharmaceutical preparation for treating epithelial tissue diseases, the pharmaceutical preparation comprising the pharmaceutical composition described in (6);

[0075] Optionally, the epithelial tissue diseases include pneumonia, enteritis, lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, prostate cancer, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gastric ulcer, duodenal ulcer, or reflux esophagitis.

[0076] Optionally, the dosage form of the pharmaceutical preparation is an intravenous injection, a local injection, a lyophilized powder for injection, a gel, a liposome-encapsulated preparation, a nanosuspension, a patch preparation, or an inhalation preparation.

[0077] In some embodiments, the intravenous injection refers to a liquid preparation prepared by aseptically suspending exosomes in physiological saline, phosphate-buffered saline (PBS) or a special cell culture medium, and is suitable for intravenous infusion and is applicable to the treatment of systemic diseases.

[0078] In some implementations, the local injection agent refers to an injectable formulation designed for specific target tissues or organs (such as tumor sites, joint cavities, abdominal cavities, etc.), which increases the concentration of exosomes at the lesion site through local administration and reduces systemic adverse reactions.

[0079] In some embodiments, the lyophilized powder injection refers to a powdered preparation made by freeze-drying an exosome suspension. It needs to be reconstituted with a solvent before use, which can improve the storage stability of exosomes and extend the shelf life. It is especially suitable for exosome drugs that need to be stored for a long time.

[0080] In some embodiments, the gel is a semi-solid formulation formed by encapsulating exosomes in a biocompatible gel matrix (such as hyaluronic acid gel, chitosan gel, etc.), suitable for local administration to the skin, mucous membranes, etc., and can slowly release exosomes to exert a long-lasting effect.

[0081] In some implementations, the liposome-encapsulated formulation refers to a composite carrier formulation formed by further encapsulating exosomes in liposomes, thereby enhancing the stability and delivery efficiency of exosomes by utilizing the targeting and biocompatibility of liposomes.

[0082] In some embodiments, the nanosuspension refers to a nanoscale suspension with uniform particle size and good stability prepared by optimizing the dispersion system of exosomes, thereby improving the circulation time and bioavailability of exosomes in vivo.

[0083] In some embodiments, the patch formulation refers to a formulation prepared by loading exosomes into pharmaceutical excipients of a transdermal patch, which is slowly released through skin penetration and is suitable for the treatment of skin diseases or local superficial tissue diseases.

[0084] In some implementations, the inhaled formulation refers to exosomes formulated as aerosols or dry powder inhalers, which are administered directly to lung lesions via the respiratory tract and are suitable for lung epithelial tissue diseases such as pneumonia and pulmonary fibrosis.

[0085] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0086] In some embodiments, the pharmaceutically acceptable excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0087] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginates, xanthan gum, and hydroxypropyl cellulose. The surfactant includes, but is not limited to, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The humectant includes, but is not limited to, glycerol and starch. The adsorbent carrier includes, but is not limited to, starch, lactose, bentonite, and soap clay. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose, and magnesium lauryl sulfate. The filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrants include, but are not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0088] In some implementations, the therapeutic agents for treating epithelial tissue diseases include, but are not limited to: anti-infective agents, anti-inflammatory agents, vitamins, cell growth factors, immunosuppressants, retinoids, or chemotherapy drugs.

[0089] Specifically, the anti-infective drugs are used to treat infectious epithelial diseases caused by bacteria, fungi, viruses, etc. Examples of such anti-infective drugs include cefuroxime, itraconazole, terbinafine, recombinant human interferon α2b, acyclovir, ribavirin, etc.

[0090] Specifically, the anti-inflammatory drugs are primarily used for non-infectious inflammatory epithelial diseases. For example, the anti-inflammatory drugs include glucocorticoids, calcineurin inhibitors, and nonsteroidal anti-inflammatory drugs (NSAIDs).

[0091] Specifically, the vitamin medication is used to treat epithelial diseases caused by vitamin deficiency. For example, the vitamin medication includes vitamin A, vitamin B, vitamin C, etc.

[0092] Specifically, the cell growth factors can promote the regeneration and repair of damaged epithelial cells. For example, the cell growth factors include epidermal growth factor, fibroblast growth factor, etc.

[0093] Specifically, the immunosuppressants are primarily targeted at autoimmune epithelial diseases. For example, the immunosuppressants include cyclophosphamide, methotrexate, etc.

[0094] Specifically, the retinoid drugs are used to treat skin keratosis disorders. Exemplarily, the retinoid drugs include retinoic acid, all-trans retinoic acid, isotretinoin, tretinoin ester, acitretin ester, acitretin, aromatic retinoic acid, ethyl aromatic retinoic acid, mesylated aromatic retinoic acid, adapalene, tazarotene (acetylretinoic acid), etc.

[0095] Specifically, the chemotherapy drugs target neoplastic epithelial diseases. Exemplarily, the chemotherapy drugs include alkylating agents such as cyclophosphamide, ifosfamide, nitrogen mustard, chlorambucil, carmustine, lomustine, semustine, thiotepa, and busulfan; antimetabolites such as methotrexate, fluorouracil, carmoflurane, tegafur, cytarabine, gemcitabine, capecitabine, and pemetrexed; and antibiotics such as actinomycin D, doxorubicin, epirubicin, pirarubicin, arubicin, and daunorubicin. Mitomycin, bleomycin, and bleomycin; plant alkaloids such as vincristine, vinblastine, vinorelbine, etoposide, teniposide, paclitaxel, docetaxel, irinotecan, and topotecan; hormones such as tamoxifen, toremifene, flutamide, bicalutamide, anastrozole, letrozole, and exemestane; and other substances such as cisplatin, carboplatin, oxaliplatin, asparaginase, dacarbazine, and temozolomide.

[0096] It should be noted that those skilled in the art can routinely select the therapeutic drugs according to the specific type of disease being treated; therefore, the present invention does not impose any particular limitation on the specific type of the therapeutic drugs.

[0097] Furthermore, the present invention also provides a method for targeted delivery of a therapeutic drug for treating epithelial tissue diseases, the method comprising the following steps: delivering the therapeutic drug using umbilical cord mesenchymal stem cell exosomes modified with an adenovirus-mediated anti-EpCAM single-chain antibody as described above.

[0098] In addition, the present invention provides a method for treating epithelial tissue diseases, the method comprising the steps of administering a therapeutically effective amount of the pharmaceutical composition or pharmaceutical preparation of the present invention as described above to a subject in need.

[0099] In this invention, the subjects include both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of this invention, the subject is a human. Subjects include patients diagnosed with epithelial tissue diseases.

[0100] In this invention, the treatment refers to delaying the development of a disease (specifically, an epithelial tissue disease as described in this invention), preventing the development of the disease, and / or reducing the severity of the symptoms that will develop or are expected to develop. Therefore, specifically, the treatment includes improving existing disease symptoms, preventing additional symptoms, improving or preventing underlying metabolic causes of symptoms, inhibiting disorders or diseases, for example, preventing the development of disorders or diseases, alleviating disorders or diseases, regressing disorders or diseases, reducing symptoms caused by diseases or disorders, or stopping the symptoms of diseases or disorders.

[0101] In this invention, the effective amount refers to the amount of a pharmaceutical composition or pharmaceutical preparation that effectively produces an ideal preventive, alleviating, or therapeutic effect. The effective amount of the pharmaceutical composition or pharmaceutical preparation described in this invention may vary depending on factors such as the pharmaceutical composition or pharmaceutical preparation, the symptoms and their severity, and the age of the mammal being treated. However, the specific dosage can be conventionally determined by those skilled in the art based on their knowledge in the field and the content disclosed in this invention. Doses capable of producing the aforementioned effects are all within the protection scope of this invention.

[0102] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may be administered in any suitable manner, including but not limited to intravenous, subcutaneous, topical, parenteral, intramuscular, oral, transdermal (which may include a penetration enhancer), and other routes of administration.

[0103] In some embodiments, the appropriate dosage of the pharmaceutical composition or pharmaceutical preparation described in this invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the desired therapeutically effective dosage, as long as it can produce the expected therapeutic and / or preventive effect on the epithelial tissue disease described in this invention. Such dosage is within the protection scope of this invention.

[0104] The fifth aspect of the invention provides for any of the following applications:

[0105] (1) The application of the adenovirus vector carrying anti-EpCAM single-chain antibody, the adenovirus carrying anti-EpCAM single-chain antibody, or the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cells described in the fourth aspect of the present invention in the preparation of a targeted delivery system for the treatment of epithelial tissue diseases.

[0106] (2) The use of the adenovirus vector carrying anti-EpCAM single-chain antibody, the adenovirus carrying anti-EpCAM single-chain antibody, or the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cells described in the fourth aspect of the present invention in the preparation of umbilical cord mesenchymal stem cell exosomes for targeted delivery of therapeutic drugs for the treatment of epithelial tissue diseases.

[0107] (3) The application of the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosomes described in the fourth aspect of the present invention in the preparation of a targeted delivery system for the treatment of epithelial tissue diseases;

[0108] (4) The use of adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosomes, or in combination with a therapeutic drug, as described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition or pharmaceutical preparation for the treatment of epithelial tissue diseases.

[0109] Furthermore, the therapeutic drug is one or more of the following: small molecule drugs, nucleic acid drugs, and protein drugs used to treat epithelial tissue diseases;

[0110] Optionally, the epithelial tissue diseases include pneumonia, enteritis, lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, prostate cancer, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gastric ulcer, duodenal ulcer, or reflux esophagitis.

[0111] In this invention, EpCAM is abnormally expressed in a variety of epithelial tissue diseases. These epithelial tissue diseases are not limited to pneumonia, enteritis, lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, prostate cancer, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gastric ulcer, duodenal ulcer, or reflux esophagitis. Any disease related to abnormal EpCAM expression will fall within the protection scope of this invention.

[0112] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0113] (1) This invention is the first to creatively construct an adenovirus-mediated anti-EpCAM single-chain antibody-modified human umbilical cord mesenchymal stem cell exosome targeted delivery system and provides a method for preparing the system. The system introduces the anti-EpCAM single-chain antibody gene into umbilical cord mesenchymal stem cells via an adenovirus vector, causing the surface of the secreted exosomes to stably express the anti-EpCAM single-chain antibody. Utilizing antigen-antibody specific binding, it achieves precise targeted delivery to epithelial tissues, effectively solving the problem of insufficient targeting of natural exosomes. Small animal imaging shows that its enrichment efficiency in the lungs and intestines is significantly higher than that of unmodified exosomes.

[0114] (2) The adenovirus Ad5 used in the targeted delivery system provided by this invention has a very high transfection efficiency (>90%) for MSCs, enabling stable expression of anti-EpCAM single-chain antibodies without additional purification steps. The preparation process is reliable and scalable, avoiding the defects of chemical coupling methods such as membrane structure damage and large batch-to-batch variations. Furthermore, comparative experiments have demonstrated that not all types of adenoviruses can effectively mediate the expression of anti-EpCAM single-chain antibodies, and the transfection efficiency of the adenovirus Ad5 used in this invention for MSCs is significantly better than that of adenoviruses Ad2 and Ad3. This result is a technical effect that would not have been anticipated by those skilled in the art based on existing technology.

[0115] (3) The targeted delivery system provided by this invention modifies exosomes with anti-EpCAM single-chain antibodies, enabling them to actively target epithelial tissues. In vivo imaging data show that the enrichment efficiency of these antibodies in the lungs and intestines is significantly higher than that of unmodified exosomes. Furthermore, the expression of anti-EpCAM single-chain antibodies transfected with adenovirus is more stable. This system is suitable for the treatment of various epithelial tissue diseases, such as pneumonia, enteritis, lung cancer, and colorectal cancer, and can simultaneously carry small molecule drugs, nucleic acids (such as siRNA), or protein drugs for synergistic treatment. In addition, human umbilical cord-derived mesenchymal stem cells better meet the requirements for clinical translation and have significant advantages in the treatment of epithelial tissue diseases, showing good application prospects and important translational significance. Attached Figure Description

[0116] Figure 1 : 4x magnification image taken with an optical microscope from MSC;

[0117] Figure 2 Diagrams identifying osteogenic, chondrogenic, and adipogenic differentiation of MSCs;

[0118] Figure 3 : Anti-Epcam adenovirus modified with Ad2, Ad3, and Ad5 vectors infected MSCs 48 h later, fluorescence microscopy image taken at 40x magnification, MOI=100, scale bar=200μm, from left to right: Ad2, Ad3, and Ad5;

[0119] Figure 4 Flowchart of the preparation of anti-Epcam-MSC-Exo;

[0120] Figure 5 Transmission electron microscope image of anti-Epcam-MSC-Exo, scale bar = 500 nm;

[0121] Figure 6 Nanoparticle tracking analysis of anti-Epcam-MSC-Exo;

[0122] Figure 7 Western blot of anti-Epcam-MSC-Exo;

[0123] Figure 8 Flow cytometry results of anti-Epcam-MSC green fluorescent protein;

[0124] Figure 9 Figure: qPCR results of anti-EpCAM single-chain antibody expression assay after MSCs were modified with adenoviruses of different MOIs;

[0125] Figure 10The early organ distribution of anti-Epcam-MSC-Exo, which binds to DiD, in C57BL / 6 mice after intravenous administration, with MSC-Exo used as a control. Detailed Implementation

[0126] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0127] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0128] Example 1: Isolation and Culture of Umbilical Cord Mesenchymal Stem Cells

[0129] 1. Experimental Materials

[0130] Primary cell source: umbilical cord tissue from donors, approved by the ethics committee and with informed consent.

[0131] Cell culture system: Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were isolated by digestion with collagenase II (Sigma-Aldrich, catalog number C7657-25MG) and hyaluronidase (Aladdin, catalog number H141272) and cultured in serum-free medium (Kelene, A1001).

[0132] Osteogenic differentiation using BI MSCgo TM Osteogenic induction differentiation medium (Israel BI, catalog number 05-440-1B), Alizarin Red S staining (Sigma, catalog number A5533); chondrogenic differentiation was performed using HyCyte. TM Chondrogenic induction medium (Noyang Bio, UBHX-D203-100) was used, and glycosaminoglycans were detected by Alcian blue staining; lipid differentiation was performed using Qida Bio lipid differentiation medium (product number P1302), and lipid droplets were observed by Oil Red O staining (Sigma).

[0133] 2. Experimental Methods

[0134] (1) Separation by enzymatic digestion: The umbilical cord tissue is cut into pieces of 1-2 mm. 3 To determine the size, digest the cells using a mixture of 0.1% collagenase II and 0.1% hyaluronidase for 30-45 minutes, then filter through a 70μm cell sieve to obtain a single-cell suspension.

[0135] (2) Culture and amplification: Serum-free medium was used for culture at 37°C and 5% CO2. The medium was changed every 3 days. When 80% confluence was reached, the medium was digested and passaged with 0.25% trypsin.

[0136] (3) Identification: Verification of osteogenic, chondrogenic and adipogenic differentiation capabilities in vitro.

[0137] 3. Experimental Results

[0138] MSC optical microscope 4x image as shown Figure 1 As shown, the basic morphological characteristics of isolated and cultured umbilical cord mesenchymal stem cells (MSCs) in an in vitro culture environment are presented. The osteogenic, chondrogenic, and adipogenic differentiation identification diagrams of MSCs are shown below. Figure 2 As shown, osteogenic differentiation revealed red mineralized nodules upon Alizarin Red S staining, chondrogenic differentiation showed a blue positive signal of glycosaminoglycans upon Alixin Blue staining, and adipogenic differentiation revealed intracellular red lipid droplets upon Oil Red O staining. These results indicate that the isolated and cultured umbilical cord mesenchymal stem cells possess multi-lineage differentiation potential, consistent with the core biological characteristics of mesenchymal stem cells.

[0139] Example 2: Construction of adenovirus vector and modification with anti-EpCAM single-chain antibody

[0140] 1. Experimental Materials

[0141] The following main reagents and materials are required for adenovirus packaging and amplification experiments: the AdMAX system (Hanheng Biotechnology, containing pHBAdMAX and pBHGlox(delta)E1,3Cre plasmids) for the construction of recombinant adenovirus.

[0142] Lipofectamine transfection reagent TM (Thermo Fisher)

[0143] HEK293 cells (ATCC) were used for packaging.

[0144] Virus purification was performed using CsCl gradient centrifugation (Sigma, catalog number C3011).

[0145] Viral titer was detected using the TCID50 method.

[0146] DMEM medium (Gibco, catalog number 11965092) and fetal bovine serum (FBS, Gibco, catalog number 10099141) are also needed for HEK293 cell culture and expansion, and 0.45μm cellulose acetate membrane (Millipore, catalog number SLHV033RS) is needed for virus filtration.

[0147] 2. Experimental Methods

[0148] (1) Adenovirus vector design: Vector systems based on Ad2, Ad3, and Ad5 adenoviruses were constructed, with the E1 / E3 region deleted in each system; sequences containing anti-EpCAM single-chain antibodies driven by the CMV promoter were inserted; and a GFP reporter gene was introduced for monitoring transfection efficiency. The same gene insertion strategy was used for the three adenovirus vectors to ensure experimental comparability.

[0149] The sequence containing the anti-EpCAM single-chain antibody is obtained by sequentially connecting the CD8 signal peptide, the anti-EpCAM single-chain antibody, the CD8 hinge region, the CD8 transmembrane domain, and customer (Δζ), and its corresponding nucleotide sequence is shown in SEQ ID NO:6;

[0150] The nucleotide sequence of the CD8 signal peptide is shown in SEQ ID NO:3; the amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO:1; the nucleotide sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO:2; the nucleotide sequence of the CD8 hinge region + CD8 transmembrane domain is shown in SEQ ID NO:4; and the nucleotide sequence of customer (Δζ) is shown in SEQ ID NO:5.

[0151] The nucleotide sequence of the adenovirus vector modified with anti-Epcam single-chain antibody (anti-Epcam adenovirus modified with Ad5 vector) constructed based on Ad5 adenovirus is shown in SEQ ID NO:7.

[0152] (2) Virus packaging and amplification: The three recombinant adenoviruses were independently packaged in HEK293 cells; purification was performed using CsCl density gradient centrifugation; and the titers of each virus type were determined (all reaching 10). 11 (PFU / mL or higher).

[0153] (3) Cell infection: Fourth-generation UC-MSCs were used for parallel infection experiments with three adenoviruses (Ad2, Ad3, and Ad5) at the same multiplicity of infection (MOI = 100). GFP expression efficiency was observed 48 hours after infection. Cell supernatants from each group were collected 72 hours later for exosome isolation. The infection conditions for the three adenoviruses were strictly controlled to be consistent throughout the experiment.

[0154] 3. Experimental Results

[0155] The image shows a fluorescence microscope image taken at 40x magnification 48 hours after MSC infection with anti-Epcam adenovirus modified with Ad2, Ad3, and Ad5 vectors. Figure 3 As shown, the results indicate that, under the same multiplicity of infection (MOI=100), the transfection efficiency or expression stability of Ad5 adenovirus on MSCs is significantly better than that of Ad2 and Ad3. This result is a technical effect that would not have been expected by those skilled in the art based on the prior art.

[0156] Furthermore, the left-hand image (results for Ad2) shows extremely weak fluorescence, indicating that Ad2 has poor transfection efficiency for MSCs and is almost ineffective in mediating fluorescent protein expression, meaning it has virtually no transfection effect. In contrast, the right-hand image (Ad5) shows significant green fluorescence, indicating that Ad5 has high transfection efficiency for MSCs and can effectively mediate fluorescent protein expression. This demonstrates that not all types of adenovirus vectors can effectively mediate fluorescent protein expression. The high transfection efficiency of Ad5 for MSCs and its ability to effectively mediate fluorescent protein expression is a technical effect that would not have been anticipated by someone skilled in the art based on existing technology.

[0157] Example 3: Isolation and Characterization of Exosomes (anti-Epcam-MSC-Exo)

[0158] 1. Experimental Materials

[0159] The following materials are required for exosome extraction by ultracentrifugation: cell culture supernatant, DPBS buffer (Gibco), ultracentrifuge (Beckman), and matching centrifuge tubes (Beckman). Transmission electron microscopy (TEM) identification of exosomes requires a copper grid and 2% phosphotungstic acid staining solution (pH 6.5). Nanoparticle tracking analysis (NTA) is performed using a NanoSight NS300 particle size analyzer (Malvern Panalytical) and its accompanying software; DPBS is used for sample dilution.

[0160] 2. Experimental Methods

[0161] (1) Separation by ultracentrifugation: Centrifugation at 300×g for 10 minutes to remove dead cells and cell debris; centrifugation at 2000×g for 10 minutes to remove microvesicles and large-diameter vesicles; ultracentrifugation at 120000×g for 90 minutes to precipitate exosomes; resuspend in PBS and filter through 0.22μm for sterilization to prepare UC-MSC exosomes (anti-Epcam-MSC-Exo) with stable expression of anti-EpCAM single-chain antibody on the surface.

[0162] (2) Characterization of exosomes. Transmission electron microscopy: 2% phosphotungstic acid negative staining was used to observe typical concave spherical structures; Nanoparticle tracking analysis: particle size distribution (typical range 70-150 nm) and concentration were determined; Western blot: expression of CD9, CD63, CD81, HSP70, TSG101 and β-actin were detected.

[0163] 3. Experimental Results

[0164] Transmission electron microscopy image of anti-Epcam-MSC-Exo as shown below Figure 5 As shown, the prepared exosomes (anti-Epcam-MSC-Exo) exhibit a typical concave spherical structure, which is consistent with the characteristic morphology of exosomes. This indicates that the product obtained by the experimental method has the typical morphological characteristics of exosomes, verifying the successful isolation of exosomes.

[0165] The nanoparticle tracking analysis diagram of anti-Epcam-MSC-Exo is shown below. Figure 6 As shown, the particle size distribution of exosomes (anti-Epcam-MSC-Exo) is presented. The typical particle size range is 70-150 nm, which is consistent with the common particle size characteristics of exosomes. This indicates that after adenovirus-mediated modification and separation, the particle size distribution of exosomes is within the normal range, and no obvious abnormal aggregation or breakage is observed.

[0166] Western blot of anti-Epcam-MSC-Exo is shown below. Figure 7 As shown, the results indicate that the exosomes express exosome marker proteins such as CD9, CD63, CD81, HSP70, and TSG101, while the expression of non-exosome marker proteins such as β-actin is as expected. This result further verifies the success of exosome preparation and shows that the modification and separation process did not affect the normal expression of exosome marker proteins, providing molecular-level evidence for the functional integrity of exosomes.

[0167] The flow cytometry results for identifying green fluorescent protein in anti-Epcam-MSCs (UC-MSCs infected with anti-Epcam adenovirus modified with Ad5 vector) are shown in the figure below. Figure 8 As shown, the expression of green fluorescent protein (GFP) in umbilical cord mesenchymal stem cells modified with adenovirus is presented. The results show that adenovirus successfully mediates the expression of the GFP gene in umbilical cord mesenchymal stem cells, verifying the effectiveness of adenovirus transfection at the cellular level.

[0168] The results of qPCR assay for anti-EpCAM single-chain antibody expression in MSCs modified with adenoviruses of different MOIs are shown in the figure below. Figure 9As shown, this demonstrates that adenovirus successfully mediates the transcription of this gene in MSCs, and the expression level shows a certain regularity with the MOI, providing data support for selecting a suitable MOI to achieve efficient and stable antibody expression, and further verifying the feasibility of the gene modification strategy.

[0169] Example 4: In vivo targeting verification

[0170] 1. Experimental Materials

[0171] Materials required for the DiD-labeled exosome tail vein injection experiment in C57BL / 6 mice include: DiD fluorescent dye (ThermoFisher, catalog number D7757) for exosome labeling (final concentration 1 μM); exosomes (200 μg / 200 μL, extracted by ultracentrifugation); male C57BL / 6 mice (6-8 weeks old) for tail vein injection; a small animal in vivo imaging system (PerkinElmerIVIS) for detecting fluorescence signals, observing the distribution of the whole body and key organs (heart, lungs, liver, spleen, kidneys, intestines) at 2, 6, 24, and 48 hours after injection; in addition, DPBS (Gibco) for diluting the exosomes, as well as an anesthetic (such as isoflurane) and dissecting instruments for in vitro imaging.

[0172] 2. Experimental Methods

[0173] (1) Exosome labeling: Exosomes modified with anti-Epcam single-chain antibody were labeled with DiD near-infrared fluorescent dye (anti-Epcam-MSC-Exo); free dye was removed by gel filtration.

[0174] (2) Animal experiments: C57BL / 6 mice (6-8 weeks old) were used; 200 μL of PBS containing 1×10⁻⁶ mg / L was injected via the tail vein. 10 DiD-labeled exosomes of the particles (DiD-bound anti-Epcam-MSC-Exo).

[0175] (3) In vivo imaging: IVIS imaging system was used to detect the tumor at 2, 6, 24 and 48 hours after injection; the main focus was on observing the fluorescence signals of the heart, lungs, liver, spleen, kidneys and intestines.

[0176] 3. Experimental Results

[0177] Early organ distribution in C57BL / 6 mice after intravenous administration of anti-Epcam-MSC-Exo, which binds to DiD, is shown in the following results. Figure 10As shown, the results indicate that the fluorescence signal in the lungs and intestines of the anti-Epcam-MSC-Exo group was significantly stronger than that of the MSC-Exo control group, demonstrating that the adenovirus-mediated modification strategy using anti-EpCAM single-chain antibodies successfully enhanced the targeting of exosomes to epithelial tissues. This result verifies the core design of this invention, "targeted delivery achieved by antigen-antibody binding between anti-EpCAM single-chain antibodies and EpCAM specifically highly expressed in epithelial cells," proving that this targeted delivery system can effectively improve the enrichment efficiency of exosomes in target organs, providing in vivo experimental evidence for its application in the treatment of epithelial tissue diseases such as pneumonia and enteritis.

Claims

1. An adenovirus-mediated modification of umbilical cord mesenchymal stem cell exosomes with anti-EpCAM single-chain antibody, characterized in that, The umbilical cord mesenchymal stem cell exosomes stably express anti-EpCAM single-chain antibody.

2. The umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that, The amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO:1; Optionally, the adenovirus is an Ad5 type adenovirus; Optionally, the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells, mouse umbilical cord mesenchymal stem cells, rat umbilical cord mesenchymal stem cells, rabbit umbilical cord mesenchymal stem cells, porcine umbilical cord mesenchymal stem cells, or canine umbilical cord mesenchymal stem cells. Optionally, the umbilical cord mesenchymal stem cell exosomes express CD9, CD63, CD81, HSP70 and / or TSG101 proteins; Optionally, the particle size of the umbilical cord mesenchymal stem cell exosomes is 70-150 nm.

3. A method for preparing adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosomes, characterized in that, The method includes the following steps: (1) Construction of adenovirus vector: Construct an adenovirus vector carrying anti-EpCAM single-chain antibody; (2) Packaging and purification: Adenovirus carrying anti-EpCAM single-chain antibody was obtained by packaging and purification; (3) Infection and harvesting: Infect umbilical cord mesenchymal stem cells, collect the cell supernatant after infection, and separate and harvest umbilical cord mesenchymal stem cell exosomes.

4. The method according to claim 3, characterized in that, The amino acid sequence of the anti-EpCAM single-chain antibody described in step (1) is shown in SEQ ID NO:1; Optionally, the adenovirus vector is an Ad5 type adenovirus vector; Optionally, the Ad5 adenovirus vector is an Ad5 adenovirus vector with the E1 / E3 region deleted; Optionally, the nucleotide sequence of the Ad5 adenovirus vector carrying the anti-EpCAM single-chain antibody is shown in SEQ ID NO:

7.

5. The method according to claim 3, characterized in that, Step (2) includes the following steps: packaging the adenovirus vector carrying the anti-EpCAM single-chain antibody in host cells and purifying it by centrifugation; Optionally, the host cell is HEK293 cell, HEK293T cell, HEK293F cell, HEK293E cell, HEK293-6E cell, HEK293FT cell, HEK293H cell, HEK293S cell or PER.C6 cell; Optionally, the centrifugation method is CsCl density gradient centrifugation, iodixanol density gradient centrifugation, or sucrose density gradient centrifugation; Optionally, the adenovirus in step (2) is an adenovirus carrying anti-EpCAM single-chain antibody obtained by packaging and purifying the adenovirus vector carrying anti-EpCAM single-chain antibody.

6. The method according to claim 3, characterized in that, Step (3) includes the following steps: infecting umbilical cord mesenchymal stem cells with the adenovirus carrying the anti-EpCAM single-chain antibody, collecting the cell supernatant after infection, and separating and harvesting umbilical cord mesenchymal stem cell exosomes; Optionally, the infection multiplicity MOI of the infection is 50-150; Optionally, the infection multiplicity MOI of the infection is 100; Optionally, the cell supernatant was collected 48-96 hours after infection; Optionally, the cell supernatant was collected 72 hours after infection; Optionally, centrifugation can be used to separate and harvest exosomes of umbilical cord mesenchymal stem cells; Optionally, the centrifugation method is ultracentrifugation; Optionally, the conditions for the ultracentrifugation method are as follows: centrifugation at (100-500)×g for 5-15 min to remove dead cells and cell debris; centrifugation at (1500-3000)×g for 5-15 min to remove microvesicles and large-diameter vesicles; and ultracentrifugation at 100000-150000×g for 60-120 min to precipitate exosomes. Optionally, the conditions for the ultracentrifugation method are as follows: centrifugation at 300×g for 10 minutes to remove dead cells and cell debris; centrifugation at 2000×g for 10 minutes to remove microvesicles and large-diameter vesicles; and ultracentrifugation at 120000×g for 90 minutes to precipitate exosomes. Optionally, step (3) further includes the following steps: after resuspending in PBS, filter and sterilize to prepare umbilical cord mesenchymal stem cell exosomes with stable surface expression of anti-EpCAM single-chain antibody; Optionally, the umbilical cord mesenchymal stem cells mentioned in step (3) are human umbilical cord mesenchymal stem cells, mouse umbilical cord mesenchymal stem cells, rat umbilical cord mesenchymal stem cells, rabbit umbilical cord mesenchymal stem cells, porcine umbilical cord mesenchymal stem cells, or canine umbilical cord mesenchymal stem cells.

7. A method for preparing adenovirus-mediated umbilical cord mesenchymal stem cells modified with anti-EpCAM single-chain antibody, characterized in that, The method includes the following steps: (1) Construction of adenovirus vector: Construct an adenovirus vector carrying anti-EpCAM single-chain antibody; (2) Packaging and purification: Adenovirus carrying anti-EpCAM single-chain antibody was obtained by packaging and purification; (3) Infection: Infect umbilical cord mesenchymal stem cells to obtain adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cells; Optionally, the amino acid sequence of the anti-EpCAM single-chain antibody described in step (1) is shown in SEQ ID NO:1; Optionally, the adenovirus vector is an Ad5 type adenovirus vector; Optionally, the Ad5 adenovirus vector is an Ad5 adenovirus vector with the E1 / E3 region deleted; Optionally, the nucleotide sequence of the Ad5 adenovirus vector carrying the anti-EpCAM single-chain antibody is shown in SEQ ID NO:7; Optionally, step (2) includes the following steps: packaging the adenovirus vector carrying the anti-EpCAM single-chain antibody in host cells and purifying it by centrifugation; Optionally, the host cell is HEK293 cell, HEK293T cell, HEK293F cell, HEK293E cell, HEK293-6E cell, HEK293FT cell, HEK293H cell, HEK293S cell or PER.C6 cell; Optionally, the centrifugation method is CsCl density gradient centrifugation, iodixanol density gradient centrifugation, or sucrose density gradient centrifugation; Optionally, the adenovirus in step (2) is an adenovirus carrying anti-EpCAM single-chain antibody obtained by packaging and purifying the adenovirus vector carrying anti-EpCAM single-chain antibody; Optionally, step (3) includes the following steps: infecting umbilical cord mesenchymal stem cells with the adenovirus carrying the anti-EpCAM single-chain antibody to obtain adenovirus-mediated anti-EpCAM single-chain antibody modified umbilical cord mesenchymal stem cells. Optionally, the infection multiplicity MOI of the infection is 50-150; Optionally, the infection multiplicity MOI of the infection is 100; Optionally, the umbilical cord mesenchymal stem cells mentioned in step (3) are human umbilical cord mesenchymal stem cells, mouse umbilical cord mesenchymal stem cells, rat umbilical cord mesenchymal stem cells, rabbit umbilical cord mesenchymal stem cells, porcine umbilical cord mesenchymal stem cells, or canine umbilical cord mesenchymal stem cells.

8. Any of the following products, characterized in that, The products include: (1) An adenovirus vector carrying an anti-EpCAM single-chain antibody, wherein the amino acid sequence of the anti-EpCAM single-chain antibody is shown in SEQ ID NO:1; Optionally, the adenovirus vector is an Ad5 type adenovirus vector; Optionally, the Ad5 adenovirus vector is an Ad5 adenovirus vector with the E1 / E3 region deleted; Optionally, the nucleotide sequence of the Ad5 adenovirus vector carrying the anti-EpCAM single-chain antibody is shown in SEQ ID NO:7; (2) An adenovirus carrying an anti-EpCAM single-chain antibody, wherein the adenovirus is an adenovirus carrying an anti-EpCAM single-chain antibody obtained by packaging and purifying the adenovirus vector carrying the anti-EpCAM single-chain antibody described in (1). (3) An adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosome prepared by any one of claims 3-6; Optionally, the umbilical cord mesenchymal stem cell exosomes stably express anti-EpCAM single-chain antibodies; Optionally, the umbilical cord mesenchymal stem cell exosomes express CD9, CD63, CD81, HSP70 and / or TSG101 proteins; Optionally, the particle size of the umbilical cord mesenchymal stem cell exosomes is 70-150 nm; (4) An adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell prepared by the method of claim 7; (5) A targeted delivery system for the treatment of epithelial tissue diseases, the targeted delivery system comprising umbilical cord mesenchymal stem cell exosomes modified with an adenovirus-mediated anti-EpCAM single-chain antibody as described in (3); Optionally, the targeted delivery system further includes a therapeutic drug carried in or on an exosome; Optionally, the therapeutic drug is one or more of small molecule drugs, nucleic acid drugs, and protein drugs used to treat epithelial tissue diseases; (6) A pharmaceutical composition for treating epithelial tissue diseases, the pharmaceutical composition comprising umbilical cord mesenchymal stem cell exosomes modified with an adenovirus-mediated anti-EpCAM single-chain antibody as described in (3); Optionally, the pharmaceutical composition further comprises a therapeutic agent for treating epithelial tissue diseases; (7) A pharmaceutical preparation for treating epithelial tissue diseases, the pharmaceutical preparation comprising the pharmaceutical composition described in (6); Optionally, the epithelial tissue diseases include pneumonia, enteritis, lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, prostate cancer, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gastric ulcer, duodenal ulcer, or reflux esophagitis. Optionally, the dosage form of the pharmaceutical preparation is an intravenous injection, a local injection, a lyophilized powder for injection, a gel, a liposome-encapsulated preparation, a nanosuspension, a patch preparation, or an inhalation preparation.

9. The following application, characterized in that, The applications include: (1) The use of the adenovirus vector carrying anti-EpCAM single-chain antibody, the adenovirus carrying anti-EpCAM single-chain antibody, or the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cells in claim 8 in the preparation of a targeted delivery system for the treatment of epithelial tissue diseases. (2) The use of the adenovirus vector carrying anti-EpCAM single-chain antibody, the adenovirus carrying anti-EpCAM single-chain antibody, or the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cells as described in claim 8 in the preparation of umbilical cord mesenchymal stem cell exosomes for targeted delivery of therapeutic drugs for the treatment of epithelial tissue diseases. (3) The use of the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosomes as described in claim 8 in the preparation of a targeted delivery system for the treatment of epithelial tissue diseases; (4) The use of the adenovirus-mediated anti-EpCAM single-chain antibody-modified umbilical cord mesenchymal stem cell exosomes as described in claim 8, or in combination with a therapeutic drug, in the preparation of a pharmaceutical composition or pharmaceutical formulation for the treatment of epithelial tissue diseases.

10. The application according to claim 9, characterized in that, The therapeutic drug is one or more of the following: small molecule drugs, nucleic acid drugs, and protein drugs used to treat epithelial tissue diseases; Optionally, the epithelial tissue diseases include pneumonia, enteritis, lung cancer, colorectal cancer, gastric cancer, breast cancer, bladder cancer, pancreatic cancer, esophageal cancer, bile duct cancer, ovarian cancer, prostate cancer, endometrial cancer, oral squamous cell carcinoma, cervical squamous cell carcinoma, gastric ulcer, duodenal ulcer, or reflux esophagitis.

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