Method for preparing intracellular-derived nanovesicles and use of nanovesicles

Abstract

Disclosed are a method for preparing intracellular-derived nanovesicles and the use of the nanovesicles, in particular the use of the nanovesicles as a drug carrier and in the treatment of ophthalmic diseases. Intracellular-derived nanovesicles can be efficiently enriched using the method. The obtained intracellular nanovesicles, compared with small extracellular vesicles that are isolated from a cell supernatant and are mainly composed of exosomes, have different lipid, protein and nucleic acid components, have a higher yield, a smaller particle size, and a narrow particle size distribution range, and are more stable at different temperatures. The intracellular nanovesicles have a higher encapsulation efficiency and drug loading rate as a drug-loading carrier, and are more easily absorbed by tissue during in vivo administration, for example, the loaded drug administered in the form of an intravitreal injection can be absorbed by the retina more quickly. The intracellular nanovesicles can not only improve corneal injuries caused by wounds and alkali burns, but can also improve retinal damage and denaturation, and have very good application and research values in the pharmaceutical field.

Description

Preparation method and application of intracellular nanovesicles Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing and applying nanovesicles derived from cells, in particular to applying the nanovesicles as drug carriers and in treating ophthalmic diseases. Background Art

[0002] To address issues such as drug solubility, biocompatibility, immunogenicity, toxic side effects, and duration of action, a wide variety of biomimetic drug carriers have been developed, including chitosan, liposomes, in vitro reconstituted lipoproteins, and biovesicles. Biovesicles, among others, possess strong solubilization capabilities and their bilayer lipid membranes offer excellent robustness and stability. As drug carriers, they offer a wide range of administration routes, high drug loading stability, high solubilization capacity, and high bioavailability. Over the past decade, numerous reports have demonstrated the use of biovesicles as drug carriers in formulations such as injections, oral suspensions or emulsions, eye drops, nasal drops, and topical dosage forms.

[0003] Cellular secretions known as extracellular vesicles (EVs), released from cells into the extracellular space or plasma and circulating in the bloodstream, have garnered extensive attention and research. EVs are primarily categorized into three types: exosomes, microvesicles or microparticles, and apoptotic bodies, with exosomes being the primary component. Exosomes are vesicles formed by two invaginations of the cell membrane. After exchanging substances within the cell, they are secreted outside the cell, participating in intercellular communication and transport of substances. They contain certain biomolecules unique to maternal cells and possess a variety of bioregulatory functions. However, exosomes suffer from limitations such as difficulty in collection, exogenous contamination, and storage, limiting their clinical translation. According to the 2018 Minimum Information for Extracellular Vesicle Research (MISEV) guidelines, it is difficult to directly prove that small particles isolated by traditional ultracentrifugation methods are purified exosomes. It is recommended to use terms such as small extracellular vesicles (sEVs) with a diameter of less than 200 nm. (Therefore, in the present invention, we use sEVs to represent small extracellular vesicles enriched by ultracentrifugation).

[0004] However, cells also contain numerous nanoscale vesicles, located between various membrane-rich organelles and responsible for intracellular transport and secretory pathways. Intracellular nanovesicles (IVs) are produced through a process known as vesicle budding and can originate from various organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes, and the plasma membrane. They are composed of a variety of membrane-rich particles, such as constitutive secretory vesicles, synaptic vesicles, COP-coated vesicles, Golgi-derived vesicles, clathrin-coated vesicles, and transport vesicles between the endoplasmic reticulum and the Golgi apparatus. These IVs are essential components of the cellular transport system, transporting substances, including proteins, RNA, and lipids, between organelles, thereby coordinating overall cellular function. Furthermore, among these IVs, secretory vesicles participate in the secretion of specific proteins, hormones, and other biomolecules through exocytosis. A subtype of secretory vesicles, also known as synaptic vesicles, plays a crucial role. Previous studies have mainly focused on the signal transduction and regulation of IVs in regulating substance transport, but there is still a lack of research and reports on how to effectively enrich and utilize these intracellular vesicles.

[0005] Stem cell therapies (such as mesenchymal stem cell therapy) show great potential in treating ophthalmic diseases (or eye diseases), particularly corneal and retinal diseases. The cornea and retina are both important components of the eye and can cause a variety of eye diseases. The cornea is the transparent, frontal portion of the outer layer of the eye, playing a vital role in transmitting light and protecting the ocular contents. Because the cornea is directly exposed to the external environment, it is susceptible to damage and infection. Sustained corneal damage can cause pain and lead to irreversible corneal opacity, resulting in blurred vision, decreased vision, and even blindness. The retina is a key part of the eye, responsible for converting light signals into electrical signals, allowing us to see images. Retinal damage or degeneration caused by genetic or light-induced factors, such as photoretinopathy, retinitis pigmentosa, macular degeneration, and hereditary retinal dystrophies, can lead to retinal cell death and loss of function, apoptosis of photoreceptor cells (including cones and rods), gradual loss of the retinal outer nuclear layer, abnormalities in the retinal pigment epithelium, and retinal dysfunction, leading to irreversible vision loss and even blindness. Stem cell therapy has been reported in animal models for the treatment of cornea and retina, but its practical application still faces numerous bottlenecks, including abnormal cell growth and immune rejection. Although extracellular vesicles secreted by stem cells share the biological properties of stem cells and can replace cells in their therapeutic effects, they still have certain drawbacks, such as low extracellular vesicle collection efficiency. After secretion, extracellular vesicles reside in the extracellular matrix, where they contain exogenous substances from the cell culture medium, resulting in reduced purity. Currently, there are no reports on the application of intracellular nanovesicles in ophthalmic diseases.

[0006] Summary of the Invention

[0007] To overcome the deficiencies of the prior art, the present invention provides a method for preparing and using intracellular nanovesicles, particularly as a drug carrier, and in the treatment of ophthalmic diseases.

[0008] In a first aspect of the present invention, a method for preparing intracellular nanovesicles is provided, which comprises the steps of sequentially subjecting cells to ultrasonic treatment, centrifugation treatment, and ultracentrifugation treatment.

[0009] Specifically, the method comprises the following steps:

[0010] (1) Dispersing cells in a suspending solvent and performing ultrasonic treatment;

[0011] (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding the cell membrane and organelle debris, and taking the supernatant;

[0012] (3) subjecting the supernatant obtained in step (2) to ultracentrifugation to obtain the precipitate as intracellular nanovesicles;

[0013] Optionally, (4) resuspending the precipitate obtained in step (3).

[0014] Specifically, the cells in step (1) are isolated cells obtained after culture, digestion, and washing (after discarding the cell culture medium, digestion, washing, etc., the possibility of obtaining extracellular vesicles by separation can be eliminated).

[0015] Specifically, the method may further include cell digestion and counting steps; in some embodiments of the present invention, the cell digestion step includes: culturing cells to 90% confluence, discarding the cell culture medium, washing the cells, adding trypsin to digest the cells, and then neutralizing and washing the cells.

[0016] Specifically, the cell density of the cells in the suspension solvent is 1-4×10 6 / mL, for example 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 In some embodiments of the present invention, the cell density is 1×10 6 pieces / mL.

[0017] Specifically, the suspending solvent is any buffer suitable for culturing cells, such as PBS, Tris buffer, or glycine buffer. In some embodiments of the present invention, the solvent is PBS.

[0018] Specifically, the cells are derived from mammals, especially humans.

[0019] Specifically, the cells are eukaryotic cells, in particular at least one of stem cells, cardiomyocytes, epithelial cells, macrophages, lymphocytes (such as T cells, B cells), tumor cells, fibroblasts, glial cells and dendritic cells; more specifically, the stem cells are adult stem cells.

[0020] Specifically, adult stem cells refer to undifferentiated cells present in a differentiated tissue. These cells are capable of self-renewal and can specialize to form cells that make up that type of tissue. Under specific conditions, adult stem cells either produce new stem cells or differentiate according to a certain procedure to form new functional cells, thereby maintaining a dynamic balance of growth and decline in tissues and organs. For example, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells (NSC), etc., especially mesenchymal stem cells.

[0021] Specifically, the adult stem cells (especially mesenchymal stem cells) are derived from mammals, especially humans.

[0022] Specifically, the cells are eukaryotic cells, in particular at least one of stem cells, cardiomyocytes, human embryonic kidney cells (293T), macrophages, T cells and tumor cells; more specifically, the stem cells are mesenchymal stem cells (MSC), including but not limited to: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid, and amniotic membrane mesenchymal stem cells.

[0023] In some embodiments of the present invention, the cells are mesenchymal stem cells, particularly umbilical cord mesenchymal stem cells (UC-MSCs).

[0024] In some embodiments of the present invention, the cells are epithelial cells, such as 293T cells, 293 / CHE-Fc cells, BHL-100 cells, BRL 3A cells, Chang cells, CHO-K1 cells, Clone 9 cells, CRFK cells, HaK cells, HK-2 cells, HTC cells, IEC-6 cells, L2 cells, LO2 cells, Vero cells, Vero-E6 cells, WI-38 cells, WISH cells, WS1 cells, etc.

[0025] In some embodiments of the present invention, the cells are lymphocytes, such as CL2 cells, CL3 cells, JAA-F11 cells, MARC S5 cells, and the like.

[0026] In some embodiments of the present invention, the cells are tumor cells, such as Hela cells, 2215 cells, A431 cells, A549 cells, AR42J cells, AtT-20 cells, B95-8 cells, bel7402 cells, BeWo cells, Caco-2 cells, Clone M-3 cells, D-17 cells, DAN cells, Daudi cells, DLD-1 cells, DSDh cells, DSN cells, Du145 cells, EB-3 cells, F9 cells, GH1 cells, GH3 cells, H9 cells, HCT-15 cells, Hela S3 cells, Hep-2 cells, Hep 3B cells, Hep G2 cells, HL-60 cells, HP cells, HT-1080 cells, HT-29 cells, HuT cells, 78 cells, HX cells, I-10 cells, IM-9 cells, JEG-3 cells, Jensen cells, Jurkat cells, K-562 cells, KB cells, LLC-WRC cells, LnCap cells, MCF7 cells, MHCC97 cells, MOLT-4 cells, NK-92 cells, PANC-1 cells, PC12 cells, Raji cells, RS4 cells, Saos-2 cells, SMMC7721 cells, SW480 cells, SW620 cells, THP-1 cells, U937 cells, U937 cells, WEHI-3 cells, XC cells, Y-1 cells, etc.

[0027] In some embodiments of the present invention, the cells are fibroblasts, such as 3T3-L1 cells, NIH3T3 cells, A9 cells, BHK-21 cells, COS-1 cells, COS-3 cells, COS-7 cells, CV-1 cells, IMR-90 cells, etc.

[0028] In some embodiments of the present invention, the cells are epithelial cells, such as 293T cells.

[0029] In some embodiments of the present invention, the cells are tumor cells, such as HeLa cells.

[0030] Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-35% (e.g., 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 34%, 35%).

[0031] Specifically, the ultrasonic treatment time in step (1) is 10-30s (e.g., 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 26, 28, 30s).

[0032] Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-25% (eg, 20%, 22%, 24%, 25%); in some embodiments of the present invention, the amplitude of the ultrasonic treatment is 20%.

[0033] Specifically, the ultrasonic treatment time in step (1) is 10-20 s (e.g., 10, 15, 18, 20), preferably 15 s; in some embodiments of the present invention, the ultrasonic treatment time is 15 s, on 2 s, off 2 s.

[0034] In some embodiments of the present invention, the number of centrifugation treatments in step (2) is two, and the respective parameters are:

[0035] 1000-3000 g (e.g., 1000, 1500, 2000, 2500, 3000 g), 5-20 minutes (e.g., 5, 8, 10, 12, 15, 20 minutes);

[0036] 10,000-30,000 g (e.g., 10,000, 15,000, 20,000, 25,000, 30,000 g), 20-40 minutes (e.g., 20, 25, 28, 30, 32, 35, 40 minutes).

[0037] In one embodiment of the present invention, the first centrifugation is performed at 2000 g for 10 minutes.

[0038] In one embodiment of the present invention, the second centrifugation is performed at 20,000 g for 30 minutes.

[0039] Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100,000-180,000 g (e.g., 100,000, 120,000, 140,000, 150,000, 160,000, 180,000), 50-100 minutes (e.g., 50, 60, 65, 70, 75, 80, 90, 100 minutes).

[0040] In one embodiment of the present invention, the ultracentrifugation is performed at 150,000 g for 70 minutes.

[0041] Specifically, the resuspension solvent in step (4) is any buffer suitable for culturing cells, such as PBS, Tris buffer, or glycine buffer. In some embodiments of the present invention, the resuspension solvent is PBS.

[0042] Specifically, one or more of the ultrasonic treatment, centrifugation and ultracentrifugation are performed at low temperature, for example, 0-5°C (e.g., 0, 1, 2, 3, 4, 5°C); in particular, the ultrasonic treatment, centrifugation and ultracentrifugation are all performed on ice; or, the ultrasonic treatment, centrifugation and ultracentrifugation are all performed at 4°C.

[0043] In some embodiments of the present invention, the method includes: taking 1×10 6 For a cell suspension with a density of 1 cell / mL, an ultrasonic probe was placed in the center of the liquid surface, and ultrasonic treatment was performed with an ultrasonic amplitude parameter range of 20% and a time parameter range of 15s, on 2s, and off 2s; the liquid was then transferred to a centrifuge tube for centrifugation with centrifugation parameters of 2000g×10min and 20000g×30min, and the supernatant was collected; the supernatant was transferred to an ultracentrifuge tube for centrifugation with centrifugation parameters of 150000g×70min.

[0044] In a second aspect of the present invention, a vesicle prepared by the method described in the first aspect is provided.

[0045] Specifically, the vesicles are small intracellular nanovesicles (sIVs).

[0046] Specifically, the vesicles are round in shape.

[0047] Specifically, the average particle size of the vesicles is 50-100 nm (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nm), especially 65-85 nm.

[0048] Specifically, the vesicles low-express exosome markers and high-express marker proteins of intracellular membrane-rich organelles and the Clathrin protein family.

[0049] Specifically, the vesicles are relatively stable at -80°C to 37°C (e.g., -80, -50, -20, -10, 0, 5, 25, 37°C).

[0050] In a third aspect of the present invention, there is provided the use of the vesicles prepared by the method of the first aspect as drug carriers for loading biomolecules in the preparation of drugs for preventing and / or treating diseases.

[0051] In a preferred embodiment of the present invention, the disease is an eye disease, in particular a retinal disease, including, but not limited to, retinal vascular diseases, such as retinal artery occlusion, retinal vein occlusion, retinal periphlebitis, diabetic retinopathy, hypertensive retinopathy, retinopathy of prematurity, etc.; macular diseases, such as central serous chorioretinopathy, age-related macular degeneration, myopic macular degeneration, cystoid macular edema, macular hole, epiretinal membrane, etc.; retinal detachment diseases, such as rhegmatogenous retinal detachment, tractional retinal detachment, exudative retinal detachment, etc.; retinitis pigmentosa; retinal tumor diseases, such as retinoblastoma, etc.; and other eye diseases, such as glaucoma, dry eye, ocular inflammatory diseases (such as uveitis, blepharitis, blepharokeratoconjunctivitis, conjunctivitis, keratitis, etc.), ocular tumors, etc.

[0052] Specifically, ocular tumors include tumors of any ocular tissue or any ocular cell, including, but not limited to, choroidal tumors, conjunctival tumors, eyelid tumors, invasive intraocular tumors, iris tumors, metastatic ocular tumors, orbital tumors, and retinal tumors. More specifically, the invention includes, but is not limited to, choroidal tumors such as choroidal hemangioma, choroidal melanoma, choroidal metastasis, choroidal nevus, choroidal osteoma, ciliary body melanoma and Ota nevus; conjunctival tumors such as conjunctival Kaposi's sarcoma, dermoid cyst on the surface of the eyeball, conjunctival lymphoma, atypical melanoma and PAM, pigmented conjunctival tumor, palpebral macules, pterygium, squamous cell carcinoma and conjunctival intraepithelial neoplasm; eyelid tumors such as basal cell carcinoma, capillary hemangioma, sweat gland cyst, palpebral nevus, seborrheic keratosis, eyelid malignant melanoma, eyelid sebaceous gland carcinoma and eyelid squamous cell carcinoma; invasive intraocular tumors such as chronic lymphocytic leukemia, Blood diseases, eyelid tumors, choroidal and intraocular lymphomas; infiltrative choroidopathy and intraocular lymphomas; iris tumors, such as anterior uveal metastases, iris cysts, iris melanocytoma, iris melanoma, and iris pearly cysts; metastatic eye tumors, such as metastatic choroidal melanoma; orbital tumors, such as adenoid cystic carcinoma of the lacrimal gland, orbital cavernous hemangioma, orbital lymphangioma, orbital mucocele, orbital inflammatory pseudotumor, orbital rhabdomyosarcoma, childhood periocular hemangioma, and sclerosing inflammatory pseudotumor; retinal tumors, such as retinal pigment epithelium (RPE) hyperplasia, retinal pigment epithelium (RPE) tumors, retinoblastoma, and von Hippel angioma.

[0053] Specifically, the vesicles serve as drug carriers to load active pharmaceutical ingredients, such as active pharmaceutical ingredients for the above-mentioned eye diseases; the active pharmaceutical ingredients include one or more of chemical drugs, biological drugs (such as proteins, peptides, oligonucleotide drugs), natural drugs, nanomedicines, radioactive drugs, photothermal therapy and photodynamic therapy drugs.

[0054] Specifically, the pharmaceutical active ingredient has a preventive and / or therapeutic effect on the disease (eg, the above-mentioned eye disease).

[0055] In some embodiments of the present invention, the chemical drugs are used for eye diseases, such as, but not limited to, aceclidine, acetazolamide, anecortave, apraclonidine, atropine, falconol, azelastine, bacitracin, benfuranolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carcoline, carteolol, celicoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cromoglycate, cyclopentolate, cyclosporine, dapagliflozin, dapoxetine ... Prazol, demecarboxamide, dexamethasone, diclofenac, dichlorosulfonamide, dipivefrin, dorzolamide, phosphocholine, emedastine, epinastine, epinephrine, erythromycin, ethoxybenzazole, eucatropine, fludrocortisone, fluorometholone, flurbiprofen, fomivirsen, neomycin B, ganciclovir, gatifloxacin, gentamicin, homatropine, hydrocortisone, idoxuridine, indomethacin, isoflurane, ketorolac, ketotifen, latanoprost, Levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medroxyprogesterone acetate, methazolamide, metipranolol, moxifloxacin, naphtholine, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, prednisolone, procaine, ranibizumab, rimidex Solanum, scopolamine, sezolamide, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, tetrahydrozoline, timolol, tobramycin, travoprost, triamcinolone, trifluridine, trimethoprim, tropicamide, unoprostone, vidarabine, xylometazoline, doxycycline, minocycline, rapamycin, curcumin, artemisinin, sorafenib, methotrexate, etc., or a combination thereof; in particular, those that are fat-soluble.

[0056] In some embodiments of the present invention, the chemical drugs used for tumors include, but are not limited to, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, doxorubicin, carmustine, lomustine, streptozotocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, capecitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, epirubicin, dapoxetine ... amycin, daunorubicin, mitoxantrone, bleomycin, mitomycin, ixabepilone, tamoxifen, flutamide, gonadorelin analogs, megestrol acetate, prednisone, dexamethasone, methylprednisolone, thalidomide, interferon alpha, folinic acid, sirolimus, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, cediranib, crenolanib, crizotinib, dabrafenib, dacomitinib nib, danusertib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib, icotinib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nilotinib, niraparib, oprozomib, olaparib, pazopanib, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, ruxolitinib, saracatinib, saridegib, sorafenib, sunitinib, tasocitinib, telatinib, tivantinib, tivozanib, tofacitinib, trametinib, vandetanib, veliparib, or a combination thereof; particularly those that are lipid-soluble.

[0057] In one embodiment of the present invention, the pharmaceutical active ingredient is rapamycin.

[0058] In other embodiments of the present invention, the active pharmaceutical ingredient is selected from the group consisting of: doxorubicin, paclitaxel, carmustine, and curcumin.

[0059] Specifically, the polypeptide and protein drugs can be, for example, cytokines (such as interleukins (such as IL-1, IL-2, IL-6, IL-10, IL-11), colony stimulating factors (such as granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF)), interferons (such as α, β, γ interferons), growth factors, tumor necrosis factors (such as TNF-α and TNF-β), transforming growth factor-β family or chemokine family, etc.) , human hemoglobin, coagulation factors, vascular endothelial growth factor antibody antagonists, protein hormones (such as insulin, glucagon, calcitonin, hypothalamic hormones, pituitary hormones or gastrointestinal hormones, etc.), antibodies (such as monoclonal antibodies, polyclonal antibodies, dimers, polymers, multispecific antibodies or antibody fragments), enzymes and coenzyme drugs (phenylalanine lyase, arginase, arginine deacylase, pancreatic ribonuclease, superoxide dismutase, asparaginase, glucocerebrosidase or hyaluronidase).

[0060] Specifically, the monoclonal antibody is selected from the group consisting of: bevacizumab, ranibizumab, aflibercept, bruceizumab, and faricizumab.

[0061] In one embodiment of the present invention, the pharmaceutical active ingredient is interleukin 10 (IL-10).

[0062] In other embodiments of the present invention, the active pharmaceutical ingredient is selected from the group consisting of bevacizumab, ranibizumab, aflibercept, bricizumab, and faricizumab.

[0063] Specifically, the drug can be administered by any suitable route, in particular intraocular administration, such as eye drops, eye ointment, subconjunctival injection, intravitreal injection, in particular intravitreal injection. Specifically, the drug can also be administered by retrobulbar injection.

[0064] Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, paste, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.

[0065] In a fourth aspect of the present invention, a method for preparing drug-loaded vesicles is provided, which comprises using the vesicles prepared by the method described in the first aspect of the present invention as drug carriers for drug loading.

[0066] Specifically, the drug loading method can be a combination of one or more methods selected from the group consisting of genetic engineering, chemical synthesis, viral vectors, ultrasound, electroporation, and co-incubation, especially ultrasound.

[0067] Specifically, the drug loading step includes: mixing the vesicles with the active pharmaceutical ingredient, incubating, ultrasonicating, and then incubating;

[0068] Optionally, free pharmaceutically active ingredient is removed (by, for example, ultrafiltration and centrifugation).

[0069] Specifically, the incubation temperature is 35-40°C (eg, 35, 36, 37, 38, 39, 40°C), especially 37°C.

[0070] Specifically, the incubation time before ultrasonic treatment is 5-20 minutes (eg, 5, 10, 15, 20 minutes), in particular 10 minutes.

[0071] Specifically, the incubation time after ultrasonic treatment is 0.5-2 hours (eg 0.5, 0.8, 1, 1.2, 1.5, 2 hours), in particular 1 hour.

[0072] Specifically, the amplitude of the ultrasonic treatment is 25%-35% (eg, 25%, 28%, 30%, 35%), especially 25%.

[0073] Specifically, the ultrasonic treatment time is 60-180 s (eg, 60, 80, 100, 120, 180 s), especially 60 s.

[0074] In some embodiments of the present invention, the ultrasonic treatment parameters include: 25% power, 6 cycles of 30 s pulse / 30 s pause.

[0075] In some embodiments of the present invention, the ultrafiltration membrane parameter is 100 kD.

[0076] Specifically, the method for preparing the drug-loaded vesicles may also include the method steps for preparing vesicles described in the first aspect of the present invention.

[0077] In a fifth aspect of the present invention, a drug-loaded vesicle is provided, wherein the vesicle prepared by the method described in the first aspect of the present invention is used as a drug carrier.

[0078] Specifically, the drug in the drug-loaded vesicles is as described in the third aspect of the present invention.

[0079] Specifically, the drug loading rate of the drug-loaded vesicles can be 40%-90% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%).

[0080] Specifically, the encapsulation efficiency of the drug-loaded vesicles can be 40%-90% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%).

[0081] In one embodiment of the present invention, the active ingredient is rapamycin; in one embodiment of the present invention, the active ingredient is interleukin 10 (IL-10).

[0082] In other embodiments of the present invention, the active pharmaceutical ingredient is selected from: doxorubicin, paclitaxel, carmustine, and curcumin; in other embodiments of the present invention, the active pharmaceutical ingredient is selected from: bevacizumab, ranibizumab, aflibercept, bruceizumab, and faricizumab.

[0083] Specifically, the preparation method of the drug-loaded vesicles can be as described in the fourth aspect of the present invention.

[0084] Specifically, the pharmaceutically active ingredient of the drug-loaded vesicles may be as described in the third aspect of the present invention.

[0085] In the sixth aspect of the present invention, a method for preventing and / or treating a disease is provided, which comprises the step of administering to a subject in need thereof a drug-loaded vesicle prepared by the method described in the first aspect (e.g., the drug-loaded vesicle described in the fourth aspect of the present invention).

[0086] In particular, the disease is preferably an eye disease, as described in the third aspect of the present invention.

[0087] Specifically, the subject is a mammal, especially a human.

[0088] Specifically, the vesicles serve as drug carriers to load active pharmaceutical ingredients, such as active pharmaceutical ingredients for the above-mentioned eye diseases; the active pharmaceutical ingredients include one or more of chemical drugs, biological drugs, natural drugs, nanomedicines, radioactive drugs, photothermal therapy drugs and photodynamic therapy drugs; in one embodiment of the present invention, the active ingredient is rapamycin; in one embodiment of the present invention, the active pharmaceutical ingredient is interleukin 10 (IL-10).

[0089] In other embodiments of the present invention, the active pharmaceutical ingredient is selected from: doxorubicin, paclitaxel, carmustine, and curcumin; in other embodiments of the present invention, the active pharmaceutical ingredient is selected from: bevacizumab, ranibizumab, aflibercept, bruceizumab, and faricizumab.

[0090] Specifically, the administration can be carried out by any suitable administration route, in particular intraocular administration, such as eye drops, eye ointment, subconjunctival injection, intravitreal injection, in particular intravitreal injection.

[0091] In a seventh aspect of the present invention, there is provided use of the vesicles described in the first aspect in the preparation of drugs for preventing and / or treating eye diseases.

[0092] In some embodiments of the present invention, the eye disease is a retinal disease, in particular a disease associated with retinal damage and / or degeneration, such as light-induced retinopathy, ischemic retinopathy, diabetic retinopathy, retinitis pigmentosa (also known as retinitis pigmentosa), cone-rod dystrophy, congenital stationary night blindness, Stargardt's disease, Leber congenital amaurosis, Best's vitelliform macular dystrophy, central macular dystrophy, Bietti crystalline corneal retinal dystrophy, Usher's syndrome, glaucoma, and age-related macular degeneration.

[0093] In some embodiments of the present invention, the eye disease is a corneal disease, such as corneal injury or corneal neovascularization.

[0094] Specifically, the cells are intracellular nanovesicles derived from adult stem cells. Adult stem cells refer to undifferentiated cells present in a differentiated tissue that are capable of self-renewal and specialization to form cells that make up that type of tissue. Under specific conditions, adult stem cells either generate new stem cells or differentiate according to a certain program to form new functional cells, thereby maintaining a dynamic balance of growth and decline in tissues and organs. For example, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), etc., in particular mesenchymal stem cells.

[0095] Specifically, the adult stem cells (especially mesenchymal stem cells) are derived from mammals, especially humans.

[0096] Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0097] In an eighth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating retinal diseases.

[0098] Specifically, the vesicle is as described in the first aspect of the present invention.

[0099] In a preferred embodiment of the present invention, the vesicles are prepared by a method comprising the following steps:

[0100] (1) Taking a mesenchymal stem cell suspension and performing ultrasonic treatment;

[0101] (2) centrifuging the liquid obtained in step (1) once or multiple times, and taking the supernatant;

[0102] (3) subjecting the supernatant obtained in step (2) to ultracentrifugation to obtain a precipitate;

[0103] Optionally, (4) resuspending the precipitate obtained in step (3).

[0104] Specifically, the mesenchymal stem cells are derived from mammals, especially humans.

[0105] Specifically, the mesenchymal stem cells include but are not limited to: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0106] In some embodiments of the present invention, the retinal disease is retinal damage and degeneration and related diseases.

[0107] Specifically, the retinal damage may be caused by various reasons, such as photochemical damage, and its manifestations may include, but are not limited to, retinal thinning, retinal detachment, retinal schisis, retinal concussion, retinal hemorrhage, exudation or ischemia.

[0108] Specifically, the retinal damage-related diseases include, but are not limited to, glaucoma, diabetic retinopathy, ischemic retinopathy, photoretinopathy, etc.

[0109] Specifically, the retinal degeneration-related diseases include, but are not limited to, retinitis pigmentosa (also known as retinitis pigmentosa), age-related macular degeneration, Stargardt's disease, cone-rod dystrophy, congenital stationary night blindness, Leber congenital amaurosis, Best's vitelliform macular dystrophy, central macular dystrophy, Bietti crystalline corneal retinal dystrophy, Usher's syndrome, etc.

[0110] Specifically, the retinal degeneration may be derived from cataracts, diabetes, glaucoma, or the like.

[0111] Specifically, the drug can be administered in any suitable manner, in particular intraocularly, such as eye drops, eye ointment, subconjunctival injection, or intravitreal injection, in particular intravitreal injection.

[0112] Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, paste, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.

[0113] A ninth aspect of the present invention provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating corneal diseases.

[0114] Specifically, the vesicle is as described in the first aspect of the present invention.

[0115] In a preferred embodiment of the present invention, the vesicles are prepared by a method comprising the following steps:

[0116] (1) Dispersing mesenchymal stem cells in a suspension solvent and performing ultrasonic treatment;

[0117] (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding the cell membrane and organelle debris, and taking the supernatant;

[0118] (3) subjecting the supernatant obtained in step (2) to ultracentrifugation, and collecting the precipitate as intracellular nanovesicles;

[0119] Optionally, (4) resuspending the precipitate obtained in step (3).

[0120] Specifically, the mesenchymal stem cells are derived from mammals, especially humans.

[0121] Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0122] Specifically, the corneal disease is selected from: keratitis (such as infectious keratitis (such as herpetic keratitis), traumatic keratitis, autoimmune keratitis), corneal ulcer (such as central corneal ulcer, Mooren's ulcer, marginal corneal ulcer, anterior chamber corneal ulcer, bacterial corneal ulcer, viral corneal ulcer, annular corneal ulcer, corneal erosion, corneal ulcer perforation), corneal perforation, superficial keratitis (such as halo keratitis, stellate keratitis, streak keratitis, nummular keratitis, photosensitive keratitis, snow blindness), electric eye, superficial punctate keratitis, filamentous keratitis, keratoconjunctivitis (such as exposure keratoconjunctivitis, nodular eye), alveolar keratoconjunctivitis, neurotrophic keratoconjunctivitis, superficial keratoconjunctivitis, exposure Corneal keratitis, stromal and deep keratitis (such as sclerosing keratitis, stromal keratitis), deep keratitis, corneal abscess, Cogan's syndrome, corneal neovascularization, corneal pannus, corneal vascular shadows, drug-induced keratoconjunctivitis, bullous keratitis, bacterial keratitis, suppurative keratitis, neuroparetic keratitis, corneal scarring and opacities, corneal scarring, corneal opacities, adhesion leukoplakia, corneal scarring, corneal clouding, corneal pannus, corneal pigmentation and deposits (such as corneal Kayser-Fleischer rings, Krukenberg spindles, Stryker lines), corneal deposits, corneal melanosis, corneal blood staining, bullous keratopathy, corneal edema, changes in corneal layers, corneal degeneration (such as corneal keratosis, keratomalacia), corneal senile rings , band keratopathy, Salzmann nodular corneal degeneration, marginal corneal degeneration, guttate keratopathy, hereditary corneal dystrophies (such as granular stromal corneal dystrophy, macular corneal dystrophy), epithelial basement membrane dystrophy, corneal dystrophies (such as Grayson-Wilbrandt corneal dystrophy, Meesmann corneal dystrophy, Schnyder corneal dystrophy, Thiel-behnke corneal dystrophy, X-linked corneal dystrophy, posterior amorphous corneal dystrophy, Descemet's corneal dystrophy, gelatinous corneal dystrophy), lattice corneal dystrophy, Fuchs corneal endothelial dystrophy, keratoconus, corneal deformities (such as corneal herniation), corneal grapes corneal swelling, Descemet's membrane bulge, corneal incision fistula, corneal inflammatory tumor, corneal epithelial damage, corneal hypoesthesia, corneal endotheliitis, corneal dissolution, corneal cyst, keratitis sicca, recurrent corneal erosion, corneal skin tag, corneal epithelial exfoliation, corneal conjunctivalization, corneal endothelial decompensation, corneal tumor, adenoviral keratoconjunctivitis, epidemic keratoconjunctivitis, corneal tumors (such as corneal epithelial carcinoma, corneal squamous cell carcinoma, corneal carcinoma in situ), corneal lesions after cataract surgery, congenital corneal opacity, congenital corneal leukoma, congenital corneal malformation (such as congenital flat cornea, congenital corneal abnormality, congenital corneal scleralization), congenital glaucoma (such as congenital spherical keratoconus with glaucoma), congenital eye malformation (such as congenital macrocornea, congenital microcornea,Congenital spherical cornea), Peter anomaly, conjunctival damage and corneal abrasion, corneal abrasion, corneal abrasion, corneal foreign body, corneal and conjunctival sac burn, corneal burn, corneal and conjunctival sac erosion, corneal erosion, corneal and conjunctival acid burn, corneal chemical burn, corneal alkaline burn, corneal acid burn, ocular tuberculosis (such as corneal tuberculosis), late congenital syphilitic eye disease (such as late congenital syphilitic keratitis, late congenital syphilitic interstitial keratitis), trachoma (such as trachomatous keratitis), herpes virus eye disease (such as herpes virus keratitis, herpes keratoconjunctivitis), herpes zoster eye disease (such as herpes zoster keratitis, herpes zoster keratoconjunctivitis), carcinoma in situ of the eye (such as corneal carcinoma in situ), ocular pemphigoid, diabetic keratopathy, iridocorneal endothelial syndrome, graft defect after corneal transplantation, Sjogren's syndrome, Stevens-Johnson syndrome, glaucoma, dry keratoconjunctivitis (such as dry eye), pink eye.

[0123] In some embodiments of the present invention, the corneal disease is keratitis, corneal ulcer, keratoconjunctivitis, or corneal tumor.

[0124] Specifically, the drug can be administered in any suitable manner, in particular, in ocular administration (eg, intraocular administration, ocular surface administration), such as eye drops, eye ointment, subconjunctival injection, intravitreal injection, in particular intravitreal injection.

[0125] Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, paste, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.

[0126] A tenth aspect of the present invention provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for repairing corneal damage.

[0127] Specifically, the intracellular nanovesicles derived from mesenchymal stem cells are as described in the first aspect of the present invention.

[0128] Specifically, the corneal damage may be corneal damage caused by various reasons, such as chemical damage, photochemical damage (such as ultraviolet damage), traumatic damage, burns (such as alkali burns), degenerative damage, ulcers, abrasions, contact lens problems, keratitis, and its manifestations include but are not limited to corneal thinning, corneal detachment, corneal splitting, corneal concussion, corneal bleeding, exudation or ischemia, etc.

[0129] In an eleventh aspect of the present invention, a method for treating eye diseases is provided, comprising the step of administering intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0130] In some embodiments of the present invention, the eye disease is a retinal disease, in particular a disease associated with retinal damage and / or degeneration, such as light-induced retinopathy, ischemic retinopathy, diabetic retinopathy, retinitis pigmentosa (also known as retinitis pigmentosa), cone-rod dystrophy, congenital stationary night blindness, Stargardt's disease, Leber congenital amaurosis, Best's vitelliform macular dystrophy, central macular dystrophy, Bietti crystalline corneal retinal dystrophy, Usher's syndrome, glaucoma, and age-related macular degeneration.

[0131] In some embodiments of the present invention, the eye disease is a corneal disease, such as corneal injury or corneal neovascularization.

[0132] Specifically, the cells are intracellular nanovesicles derived from adult stem cells. Adult stem cells refer to undifferentiated cells present in a differentiated tissue that are capable of self-renewal and specialization to form cells that make up that type of tissue. Under specific conditions, adult stem cells either generate new stem cells or differentiate according to a certain program to form new functional cells, thereby maintaining a dynamic balance of growth and decline in tissues and organs. For example, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), etc., in particular mesenchymal stem cells.

[0133] Specifically, the adult stem cells (especially mesenchymal stem cells) are derived from mammals, especially humans.

[0134] Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0135] In a twelfth aspect of the present invention, a method for preventing and / or treating retinal diseases is provided, comprising the step of administering mesenchymal stem cell-derived nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0136] Specifically, the disease is as described in the first aspect of the present invention.

[0137] Specifically, the subject is a mammal, especially a human.

[0138] Specifically, the administration can be carried out by any suitable administration method, in particular intraocular administration, such as eye drops, eye ointment, subconjunctival injection, and intravitreal injection, in particular intravitreal injection and eye drops.

[0139] In some embodiments of the present invention, the administration method is intravitreal injection, and the administration amount can be 1 μg-30 μg vesicles / eye.

[0140] In a thirteenth aspect, the present invention provides a method for preventing and / or treating corneal diseases, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0141] Specifically, the disease is as described in the first aspect of the present invention.

[0142] Specifically, the subject is a mammal, especially a human.

[0143] Specifically, the administration can be carried out by any suitable route, in particular ocular administration (e.g., intraocular administration, ocular surface administration), such as eye drops, eye ointment, subconjunctival injection, and intravitreal injection, in particular intravitreal injection and eye drops.

[0144] In some embodiments of the present invention, the administration method is intravitreal injection, and the administration amount can be 1 μg-30 μg vesicles / eye.

[0145] The present invention has the following beneficial effects:

[0146] (1) The present invention provides a method for collecting intracellular nanovesicles by ultrasonic cell disruption, which can avoid the tedious steps and impurities of collecting extracellular vesicles, that is, the operation of separating vesicles is simple, and the yield of the obtained vesicles is high. The obtained small intracellular nanovesicles have a smaller particle size, a narrower particle size distribution range, and are more stable at different temperatures compared to extracellular vesicles with exosomes as the main component. The obtained intracellular nanovesicles have good tissue compatibility. Compared with extracellular vesicles, the intracellular nanovesicles have a wider range and degree of distribution when injected in situ into the tissue. When used as a carrier to load drugs, they have a higher encapsulation rate and drug loading rate. For example, in the form of intravitreal injection, the drugs loaded by the intracellular nanovesicles can be absorbed faster by the retina. Therefore, the nanovesicles of the present invention are highly customizable and can be flexibly loaded with different types of drugs, growth factors, antibodies or gene therapy fragments according to the needs of specific ophthalmic diseases, so as to realize the formulation of personalized treatment plans. This not only broadens the treatment methods for ophthalmic diseases, but also lays a solid foundation for exploring more accurate and efficient ophthalmic treatment strategies in the future.

[0147] (2) The small intracellular nanovesicles prepared by the present invention have very good application and research value in ophthalmic diseases (especially corneal diseases and retinal diseases). They can not only improve corneal damage caused by trauma and alkali burns, promote corneal epithelial and stroma repair, promote corneal sensitivity recovery, reduce corneal opacity, conjunctival congestion, and conjunctival edema, and reduce corneal neovascularization and conjunctival secretion production, but also improve retinal damage and degeneration, slow down retinal thinning, reduce retinal cell apoptosis, reduce retinal photoreceptor cell death, reduce retinal inflammation, and inhibit retinal endoplasmic reticulum stress. They have very good application and research value in the medical field (especially in the treatment of eye diseases). BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 shows a schematic diagram of the process and steps of the method for producing intracellular nanovesicles, wherein A shows the process diagram and B shows the steps.

[0149] Figure 2 shows the optimization process for the isolation parameters of intracellular nanovesicles. Figures 2A and 2B show the protein yield (Figure 2A) and vesicle yield (Figure 2B) of sIVs obtained at different sonication times and an ultrasonic amplitude of 20%. At a sonication amplitude of 20%, the vesicle yield dropped sharply when the sonication time exceeded 10 seconds or exceeded 20 seconds. Figures 2C and 2D show the protein yield (Figure 2C) and vesicle yield (Figure 2D) of sIVs obtained at different sonication times and an ultrasonic amplitude of 15 seconds. At a sonication time of 15 seconds, the vesicle yield dropped sharply when the sonication amplitude exceeded 25%. Figure 2E shows transmission electron micrographs of sIVs obtained at different sonication times and an ultrasonic amplitude of 20%. Scale bar: 100 nm. Figure 2F shows transmission electron micrographs of sIVs obtained at different sonication times and an ultrasonic amplitude of 15 seconds. Scale bar: 100 nm.

[0150] Figure 3 shows transmission electron micrographs of sEVs and sIVs from MSCs (A), 293T (B), and Hela (C) cells, respectively. Scale bar for wide-field images: 200 nm; scale bar for close-up images: 200 nm.

[0151] Figure 4 shows the results of nanoparticle size analysis, which shows the particle size distribution of MSCs (A), 293T (B), Hela (C) cells and their sEVs and sIVs.

[0152] Figure 5 shows the statistical analysis results of the particle sizes of MSCs, 293T cells, Hela cells, and their sEVs and sIVs (**p < 0.01, ***p < 0.001 indicate significant differences among the groups).

[0153] Figure 6 shows the statistical analysis results of the number of sEVs and sIVs vesicles (A, B, C) and total protein production (D, E, F) of MSCs, 293T, and Hela cells with equal cell numbers (**p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant differences between the groups).

[0154] FIG7 shows the results of Coomassie Brilliant Blue staining, which shows the protein distribution of MSCs (A), 293T (B), Hela (C) cells and their sEVs and sIVs.

[0155] Figure 8 shows the Western blot results, which show the expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD81) in MSCs (A), 293T (B), and Hela (C) cells.

[0156] Figure 9 shows transmission electron micrographs of sEVs and sIVs from MSCs (A), 293T (B), and Hela (C) at different temperatures. Scale bar: 200 nm.

[0157] Figure 10 shows the results of nanoparticle size analysis of sEVs and sIVs from MSCs, 293T cells, and Hela cells at different temperatures (-80°C, 4°C, and 37°C). Figure 10A shows the particle size distribution, and Figure 10B shows the statistical analysis of particle size (*p < 0.05, **p < 0.01 indicate significant differences between groups).

[0158] Figure 11 shows the results of proteomic analysis, which revealed distinct protein expression profiles of sIVs from MSCs, 293T cells, and HeLa cells compared to sEVs. The large circle in the upper left corner represents proteins uniquely expressed by sIVs from MSCs, distinct from sEVs; the large circle in the upper left corner represents proteins uniquely expressed by sIVs from 293T cells, distinct from sEVs; the large circle below represents proteins uniquely expressed by sIVs from HeLa cells, distinct from sEVs; and the overlapping area in the middle represents proteins uniquely expressed by sIVs from all three cell types, totaling 106 proteins.

[0159] FIG12 shows the co-expressed sIVs-specific proteins in MSCs (A), 293T (B) and Hela cells (C), arranged from high to low abundance, showing the top 50 most highly expressed proteins of each.

[0160] Figure 13 shows the results of super-resolution microscopy and total internal reflection fluorescence structured illumination microscopy. Among them, Figure 13A shows the TRIF-SIM mode (showing cell membrane), which shows that there are CD63-positive areas on the cell membrane surface (left picture), while almost no TMEM214-positive areas are observed (right picture). Figure 13B shows the Widefield-2DSM mode (showing whole cell), which proves that CD63-positive (left picture) and TMEM214-positive (right picture) signals are present in the whole cell. Figure 13C shows time-lapse screenshots of dynamic observation of living cells. The left picture shows CD63-labeled late endosomes, sEVs and cell membranes in the cell. The arrow at 0s shows that sEVs have just been released from the cell membrane to the outside of the cell, and gradually move away from the cell membrane from 6min to 14min. The right picture shows TMEM214, which shows that sIVs are diffusely distributed in the cell and are not released outside the cell.

[0161] FIG14 shows a Venn diagram showing the total protein species in MSCs (A), 293T (B) and Hela (C) cells, sEVs, and sIVs.

[0162] FIG15 shows the principal component analysis results of total proteins identified in MSCs (A), 293T (B) and Hela (C) cells, sEVs, and sIVs.

[0163] FIG16 shows a heat map showing differentially expressed proteins between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0164] FIG17 shows a volcano plot showing the top five significantly differentially expressed proteins between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0165] FIG18 shows a heat map showing the differential expression of exosomal markers between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0166] FIG19 shows heat maps showing the differential expression of organelle markers between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0167] FIG20 shows a heat map showing the differential expression of Clathrin family proteins between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0168] FIG21 shows the cellular component enrichment analysis of sIVs co-expressed proteins in MSCs, 293T cells, and Hela cells.

[0169] FIG22 shows the biological process enrichment analysis of sIVs co-expressed proteins in MSCs, 293T and Hela.

[0170] Figure 23 shows the differences in IL-1β and IGF2 cytokine levels between sEVs and sIVs of MSCs detected by protein profiling. Figure 23A shows the differences in IL-1β cytokine levels between sEVs and sIVs of MSCs detected by protein profiling, and Figure 23B shows the differences in IGF2 cytokine levels between sEVs and sIVs of MSCs detected by protein profiling (*p < 0.05, ***p < 0.001 indicate significant differences between the groups).

[0171] Figure 24 shows the differences in the levels of IGF-1, EGF, IL-10, IL-6, and TNFα cytokines carried by sEVs and sIVs of MSCs detected by ELISA. Figure 24A shows the differences in the levels of IGF-1 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 24B shows the differences in the levels of EGF cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 24C shows the differences in the levels of IL-10 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 24D shows the differences in the levels of IL-6 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, and Figure 24E shows the differences in the levels of TNFα cytokines carried by sEVs and sIVs of MSCs detected by ELISA (**p < 0.01, ***p < 0.001 indicate significant differences between the groups, ns indicates no statistical difference).

[0172] Figure 25 shows the relative RNA abundance in sEVs and sIVs of MSCs, 293T and Hela cells (ns indicates no statistical difference).

[0173] Figure 26 shows the percentage of small noncoding RNAs read from small RNAs in sEVs and sIVs from MSCs (A), 293T (B), and HeLa (C) cells. miRNA: micro-RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; tRNA: transfer RNA; rRNA: ribosomal RNA.

[0174] FIG27 is a Venn diagram showing the miRNA species contained in sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0175] FIG28 shows the principal component analysis results of MSCs (A), 293T (B) and Hela (C) cell miRNA datasets.

[0176] FIG29 shows a matchstick chart showing the top 10 highly abundant miRNAs in sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

[0177] FIG30 shows a heat map showing that there are many differentially expressed miRNAs between sEVs and sIVs of MSCs (A), 293T (B) and Hela (C) cells.

[0178] Figure 31 shows a volcano plot showing the top five differentially expressed miRNAs between sEVs and sIVs of MSCs (A), 293T (B), and HeLa (C) cells. The abscissa represents the fold change (log2 fold difference) of miRNA expression between different samples or comparison combinations, and the ordinate represents the significance level of the expression difference.

[0179] Figure 32 shows enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from MSCs. A shows the top 10 terms in GO analysis for biological process (BP), cellular component (CC), and molecular function (MF). B shows KEGG enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from MSCs.

[0180] Figure 33 shows enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from 293T cells. A shows the top 10 terms in GO analysis for biological process (BP), cellular component (CC), and molecular function (MF). B shows KEGG enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from 293T cells.

[0181] Figure 34 shows enrichment analysis of candidate target genes of differentially expressed miRNAs in Hela cell-derived sEVs and sIVs. A shows the top 10 entries in GO analysis for biological process (BP), cellular component (CC), and molecular function (MF). B shows KEGG enrichment analysis of candidate target genes of differentially expressed miRNAs in Hela cell-derived sEVs and sIVs.

[0182] Figure 35 shows the types and proportions of metabolites contained in sEVs and sIVs of MSCs (A), 293T (B) and Hela cells (C).

[0183] Figure 36 shows the principal component analysis results of lipids contained in sEVs and sIVs of MSCs (A), 293T (B) and Hela cells (C).

[0184] FIG37 shows a heat map showing the differential lipid types contained in sEVs and sIVs of MSCs (A), 293T (B), and HeLa cells (C).

[0185] Figure 38 shows lipidome histograms for MSCs (A), 293T (B), and HeLa (C) cells in the sIVs group versus the sEVs group. The horizontal axis represents the relative percentage change in the content of each substance in the comparison group. A relative percentage change of zero indicates that the substance is present at the same level in both groups; a positive relative percentage change indicates that the substance is present at a higher level in the sIVs group; a negative relative percentage change indicates that the substance is present at a higher level in the sEVs group. The vertical axis of the lipidome histogram represents lipid classification information.

[0186] Figure 39 shows the results of an investigation into the ability of intracellular nanovesicles to be endocytosed by RPE cells cultured in vitro. Figures 39A and B show that DiD-labeled sEVs and sIVs were co-incubated with RPE cells for 3 h, 12 h, 24 h, and 48 h. The first row of figures shows the cytoskeleton, the second row of figures shows the vesicles, and the third row of figures shows a composite of the cytoskeleton, vesicles, and nuclei. Scale bar: 20 μm. Figure 39C shows the statistical results of DiD fluorescence intensity in the cells (*p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups).

[0187] Figure 40 shows the results of an investigation into the ability of intracellular nanovesicles to be endocytosed by HRMECs cultured in vitro. Figures 40A and B show DiD-labeled sEVs and sIVs co-incubated with HRMECs for 3 h, 12 h, 24 h, and 48 h. The first row of images shows the cytoskeleton, the second row of images shows the vesicles, and the third row of images shows a composite image of the cytoskeleton, vesicles, and nucleus. Scale bar: 20 μm. Figure 40C shows the statistical results of DiD fluorescence intensity within the cells (*p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups).

[0188] Figure 41 shows the results of an investigation into the ability of intracellular nanovesicles to be internalized by the retina. Figure 41A shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 24h and 48h after subconjunctival injection. Figure 41B shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 8h and 24h after intravitreal injection. DAPI staining shows the cell nucleus. Scale bar: 20μm. Figure 41C shows the statistical results of DiD fluorescence intensity in the retina of the subconjunctival injection group. Figure 41D shows the statistical results of DiD fluorescence intensity in the retina of the intravitreal injection group (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate significant differences between the groups).

[0189] Figure 42 shows the results of the drug-loading performance of intracellular nanovesicles. Figure 42A shows transmission electron microscopy images of vesicle morphology under natural and ultrasonic loading conditions. Figure 42B shows the nanoparticle size analysis results of vesicles under natural and ultrasonic loading conditions. Scale bar: 200 nm.

[0190] Figure 43 shows the results of the drug loading performance test of intracellular nanovesicles. Among them, Figure 43A shows the chromatographic peaks of Rapa in Rapa sEVs and Rapa sIVs detected by high-performance liquid chromatography, characterized by a clear large peak and a small peak. Figure 43B shows the effect of sEVs and sIVs on Rapa encapsulation efficiency (n=6). Figure 43C shows the effect of sEVs and sIVs on Rapa drug loading efficiency (***p<0.001 indicates significant difference between groups).

[0191] Figure 44 shows the results of the drug loading performance test of intracellular nanovesicles. Figure 44A shows the retinal Rapa content at different times after subconjunctival injection of Rapa, sEVs-Rapa, and sIVs-Rapa. Figure 44B shows the retinal Rapa content 24 hours after intravitreal injection of Rapa, sEVs-Rapa, and sIVs-Rapa (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant differences between the groups).

[0192] FIG45 shows the drug loading efficiency of IL10 encapsulated in sIVs.

[0193] FIG46 shows the results of T cell proliferation detection by flow cytometry.

[0194] Figure 47 shows the statistical results of T cell proliferation detected by flow cytometry. **p<0.01 indicates significant difference between the groups.

[0195] Figure 48 shows that MSC-sIVs protect retinal thickness from blue light damage. Among them, Figure 48A shows a representative heat map showing the thickness of the retina. Figure 48B shows a representative SD-OCT scan of the retina. Figure 48C shows the statistical results of the average thickness of the retina, where BL represents light damage model mice, L is the low-dose administration group, and H is the high-dose administration group (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate statistical differences between the groups).

[0196] Figure 49 shows that MSC-sIVs protect retinal outer nuclear layer cells from blue light-induced damage. Figure 49A shows representative H&E-stained retinal images of each group (GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). Black scale bar: 100 μm; white scale bar: 20 μm. Figure 49B shows the counting of ONL nuclei in eight areas of retinal sections one week after blue light induction, starting from 250 μm from the optic nerve head and extending outward every 250 μm or 500 μm along the dorsal / superior edge (positive values ​​on the horizontal axis) and ventral periphery / inferior edge (negative values ​​on the horizontal axis), where BL represents light-damaged model mice, L represents the low-dose administration group, and H represents the high-dose administration group (compared with BL-PBS, the statistical differences between each group are indicated by letters, a represents NoBL-PBS, b represents BL-sIVs-H, c represents BL-sEVs-H, d represents the statistical difference between BL-sEVs-H and BL-sIVs-H, 1 letter p < 0.05, 2 letters p < 0.01, 3 letters p < 0.001 and 4 letters p < 0.0001 indicate significant differences between the groups).

[0197] Figure 50 shows that MSC-sIVs protect retinal electrophysiological function from blue light damage. Figure 50A shows a representative ERG waveform [2.2Log (cd.s / m2)] from one eye of a single mouse in each treatment group under a stimulating flash. Figure 50B shows the average amplitude of the ERG a-wave and b-wave in each treatment group, where BL represents the light-damaged model mouse (compared with BL-PBS, the statistical differences between the groups are indicated by letters; a represents BL-sIVs-H, b represents BL-sEVs-H, c represents NoBL-PBS, and d shows the statistical difference between BL-sEVs and BL-sIVs, with 1 letter p < 0.05, 2 letters p < 0.01, 3 letters p < 0.001 and 4 letters p < 0.0001 indicating significant differences between the groups).

[0198] Figure 51 shows that MSC-sIVs reduce blue light-induced retinal apoptosis. Figure 51A shows representative morphological images of each group; the signal in the first row of figures is TUNEL-positive, and the signal in the second row of figures other than the TUNEL-positive signal is DAPI-stained cell nucleus. Scale bar: 20 μm. Figure 51B shows the TUNEL-positive cell nuclei counted from the entire retina, where BL represents light-damaged model mice, L is the low-dose administration group, and H is the high-dose administration group (**p<0.01, ***p<0.0001 indicate statistical differences from the NoBL-PBS group. #### p<0.0001 indicates statistical difference compared with the BL-PBS group).

[0199] Figure 52 shows that MSC-sIVs reduce blue light-induced photoreceptor cell damage. Figure 52A shows the expression of rhodopsin protein in the mouse retina measured by immunofluorescence. The cell nuclei were stained with DAPI. Scale bar: 20 μm. Figure 52B shows the relative expression of rhodopsin protein in the retina, where BL represents light-damaged model mice, L is the low-dose administration group, and H is the high-dose administration group (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistical differences compared with the NoBL-PBS group; ## p<0.01, #### p < 0.0001 indicated statistical difference compared with the BL-PBS group; & p<0.05, &&&& p<0.0001 indicates statistical difference between sEVs and sIVs of the same dose).

[0200] Figure 53 shows the Western blot analysis of GFAP and rhodopsin in the retina. Figure 53A shows the Western blot analysis of GFAP and rhodopsin in the mouse retina. Figure 53B shows the quantitative analysis of GFAP, and Figure 53C shows the quantitative analysis of rhodopsin. BL represents light-damaged model mice, L is the low-dose administration group, and H is the high-dose administration group (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate statistical differences compared to the BL-PBS group).

[0201] Figure 54 shows that MSC-sIVs inhibit blue light-induced retinal Müller cell activation. Among them, Figure 54A shows the immunofluorescence method used to determine the expression of GFAP in the mouse retina. The cell nucleus was stained with DAPI. Scale bar: 20μm. Figure 54B shows the relative expression of GFAP in the retina, where BL represents the light damage model mouse, L is the low-dose administration group, and H is the high-dose administration group (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistical differences compared with the NoBL-PBS group, # p < 0.05 and ## p<0.01 indicates statistically significant difference compared with the BL-PBS group).

[0202] Figure 55 shows that MSC-sIVs inhibits the increase in the number of retinal microglia induced by blue light. Among them, Figure 55A shows the distribution of Iba1-positive cells in the retina. Scale bar: 20μm. Figure 55B shows the quantitative analysis of Iba1-positive cells in the entire retina. Figure 55C shows the quantitative analysis of Iba1-positive cells in the inner retina. Figure 55D shows the quantitative analysis of Iba1-positive cells in the outer retina, where BL represents light-damaged model mice, L is the low-dose administration group, and H is the high-dose administration group (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate significant differences from the NoBL-PBS group, # p<0.05, ## p<0.01, ### p<0.001 and #### p<0.0001 indicated a significant difference compared with the BL-PBS group. & p < 0.05 and && p<0.01 indicates significant differences among groups at the same dose).

[0203] Figure 56 shows that MSC-sIVs slowed the loss of outer nuclear layer cells in rd10 mice. Figure 56A shows representative H&E-stained retinal images of each group (GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). Figure 56B shows the counting of ONL nuclei in eight discrete fields of retinal sections, starting at 250 μm from the optic nerve head and extending outward every 250 μm or 500 μm along the dorsal / superior edge (positive values ​​on the horizontal axis) and ventral periphery / inferior edge (negative values ​​on the horizontal axis) (compared with rd10-PBS, statistical differences between each group are indicated by letters, a represents rd10-sIVs, b represents rd10-sEVs; c represents the comparison between rd10-sEVs and rd10-sIVs, 1 letter p < 0.05, 2 letters p < 0.01, 3 letters p < 0.001 and 4 letters p < 0.0001 indicate significant differences between groups).

[0204] Figure 57 shows that MSC-sIVs rescue retinal electrophysiological function in rd10 mice. Figure 57A shows representative ERG waveforms from one eye of a single mouse in each treatment group at p28 in rd10 mice [2.2Log(cd.s / m2)]. Figure 57B shows the average amplitudes of the ERG a-wave and b-wave in each treatment group (a indicates the statistical difference between rd10-sIVs and rd10-PBS, b indicates the statistical difference between rd10-sEVs and rd10-PBS, and c indicates the statistical difference between rd10-sEVs and rd10-sIVs. 1 letter p < 0.05, 2 letters p < 0.01, 3 letters p < 0.001, and 4 letters p < 0.0001).

[0205] Figure 58 shows that MSC-sIVs reduce retinal apoptosis in rd10 mice. Figure 58A shows representative morphological images of each group; the signal in the first row is TUNEL, and the signal in the second row, other than TUNEL, is DAPI-stained cell nuclei. Scale bar: 20 μm. Figure 58B shows the enumeration of TUNEL-positive nuclei in the ONL from whole retinas (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate significant differences between groups).

[0206] Figure 59 shows that MSC-sIVs rescue retinal photoreceptor cell loss in rd10 mice. Figure 59A shows the expression of rhodopsin protein in the mouse retina measured by immunofluorescence. Cell nuclei were stained with DAPI. Scale bar: 20 μm. Figure 59B shows the relative expression of rhodopsin protein in the retina by fluorescence quantitative analysis (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate significant differences between groups).

[0207] Figure 60 shows that MSC-sIVs rescue retinal synaptic loss in rd10 mice. Figure 60A shows PSD95 protein expression in mouse retinas measured by immunofluorescence. Cell nuclei were stained with DAPI. Scale bar: 20 μm. Figure 60B shows the relative expression of PSD95 protein in the retina by fluorescence quantitative analysis (*p<0.05, **p<0.01, and ****p<0.0001 indicate significant differences between groups).

[0208] Figure 61 shows that MSC-sIVs reduce Müller cell activation in rd10 mice. Figure 61A shows immunofluorescence analysis of GFAP expression in the mouse retina. Cell nuclei were stained with DAPI. Scale bar: 20 μm. Figure 61B shows the relative expression of GFAP in the retina (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significant differences between groups).

[0209] Figure 62 shows that MSC-sIVs reduce retinal microglial proliferation in rd10 mice. Figure 62A shows the distribution of Iba1-positive cells in the retina. Scale bar: 20 μm. Figure 62B shows the quantitative analysis of Iba1-positive cells in the whole retina, inner retina, and outer retina (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate significant differences between groups).

[0210] Figure 63 shows the distribution of microglia in retinal sections using immunofluorescence staining of Iba1. Scale bar: 20 μm.

[0211] Figure 64 shows Western blot analysis of GFAP, PSD95, and rhodopsin in the retina. Figures 64A and 64B show Western blot analysis and quantitative analysis of GFAP, PSD95, and rhodopsin in the mouse retina, respectively (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate significant differences between groups).

[0212] Figure 65 shows the differential gene pathway enrichment analysis of MSC-sIVs compared with PBS in the retina of light-damaged mice.

[0213] FIG66 shows the differential gene pathway enrichment analysis of MSC-sIVs compared with PBS in the rd10 mouse retina.

[0214] Figure 67 shows the heat map of GSVA enrichment analysis of total proteins in MSC-sEVs and MSC-sIVs.

[0215] Figure 68 shows the expression of the top 50 miRNAs in terms of total abundance in MSC-sEVs and MSC-sIVs.

[0216] Figure 69 shows the differential enrichment analysis of the top 50 miRNA target genes in terms of total abundance in MSC-sEVs and MSC-sIVs.

[0217] Figure 70 shows the expression of endoplasmic reticulum stress marker proteins at different time points after blue light irradiation of mouse retinas. Figure 70A shows the expression of GRP78 protein in mouse retina detected by Western blot, using β-actin as an internal control. Figure 70B shows the statistical results for GRP78. (n=6, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicate statistically significant differences compared to the NoBL-PBS group).

[0218] Figure 71 shows that MSC-sIVs inhibited endoplasmic reticulum stress in the retina of mice damaged by blue light. Figure 71A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 71B shows the statistical results of GRP78. Figure 71C shows the statistical results of p-PERK / PERK. Figure 71D shows the statistical results of p-PERK. Figure 71E shows the statistical results of PERK. Figure 71F shows the statistical results of IRE1a. Figure 71G shows the statistical results of ATF6. Figure 71H shows the statistical results of p-eIF2a / eIF2a. Figure 71I shows the statistical results of p-eIF2a. Figure 71J shows the statistical results of eIF2a. Figure 71K shows the statistical results of CHOP. (n=4-6, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistically significant differences compared with the NoBL-PBS group, # p<0.05, ## p<0.01, ### p<0.001 and ####p<0.0001 indicates a statistically significant difference compared with the BL-PBS group. & p<0.05, && p<0.01, &&& p<0.001 and &&&& p<0.0001 indicates statistical difference between sEVs and sIVs of the same dose).

[0219] Figure 72 shows that MSC-sIVs inhibited the apoptosis signal of the retina of mice damaged by blue light. Among them, Figure 72A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 72B shows the statistical results of Cleaved-Caspase3 / -Caspase3. Figure 72C shows the statistical results of Caspase3. Figure 72D shows the statistical results of Bax. Figure 72E shows the statistical results of Bcl-2. (n=4-6, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistical differences compared with the NoBL-PBS group, # p<0.05, ## p<0.01, ### p<0.001 and #### p<0.0001 indicates a statistically significant difference compared with the BL-PBS group. & p<0.05, && p<0.01, &&& p<0.001 and &&&& p<0.0001 indicates statistical difference between sEVs and sIVs of the same dose).

[0220] Figure 73 shows the expression of endoplasmic reticulum stress marker proteins in the retina of rd10 mice at different time points. Figure 73A shows the expression of GRP78 protein in the mouse retina by Western blot, using β-actin as an internal control. Figure 73B shows the statistical results for GRP78. (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant differences between the two groups.)

[0221] Figure 74 shows that MSC-sIVs inhibit endoplasmic reticulum stress in the p21 retina of rd10 mice. Figure 74A shows a schematic diagram of target protein expression in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 74B shows the statistical results for GRP78. Figure 74C shows the statistical results for p-PERK / PERK. Figure 74D shows the statistical results for p-PERK. Figure 74E shows the statistical results for PERK. Figure 74F shows the statistical results for IRE1a. Figure 74G shows the statistical results for ATF6. Figure 74H shows the statistical results for p-eIF2a / eIF2a. Figure 74I shows the statistical results for p-eIF2a. Figure 74J shows the statistical results for eIF2a. Figure 74K shows the statistical results for CHOP. (n=4-6, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistical differences between the two groups).

[0222] Figure 75 shows that MSC-sIVs inhibit apoptosis signaling in the p21 retina of rd10 mice. Figure 75A shows a schematic diagram of target protein expression in the mouse retina detected by Western blot, with β-actin used as an internal control. Figure 75B shows the statistical results for cleaved-Caspase3 / -Caspase3. Figure 75C shows the statistical results for Caspase3. Figure 75D shows the statistical results for Bax. Figure 75E shows the statistical results for Bcl-2. (n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant differences between the two groups.)

[0223] Figure 76 shows the inhibition of retinal endoplasmic reticulum stress in rd10 mice p28 by MSC-sIVs. Figure 76A shows a schematic diagram of target protein expression in mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 76B shows the statistical results for GRP78. Figure 76C shows the statistical results for p-PERK / PERK. Figure 76D shows the statistical results for p-PERK. Figure 76E shows the statistical results for PERK. Figure 76F shows the statistical results for IRE1a. Figure 76G shows the statistical results for ATF6. Figure 76H shows the statistical results for p-eIF2a / eIF2a. Figure 76I shows the statistical results for p-eIF2a. Figure 76J shows the statistical results for eIF2a. Figure 76K shows the statistical results for CHOP. (n=4-6, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate statistical differences between the two groups).

[0224] Figure 77 shows that MSC-sIVs inhibit apoptotic signaling in the p28 retina of rd10 mice. Figure 77A shows a schematic diagram of target protein expression in mouse retina detected by Western blot, with β-actin used as an internal control. Figure 77B shows the statistical results for cleaved-Caspase3 / -Caspase3. Figure 77C shows the statistical results for Caspase3. Figure 77D shows the statistical results for Bax. Figure 77E shows the statistical results for Bcl-2. (n = 3-6, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate statistically significant differences between the two groups.)

[0225] Figure 78 shows the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 24 hours. The control group was treated with PBS. * indicates the statistical difference between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates the difference between sIVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0226] Figure 79 shows a line graph showing the effects of different concentration gradients of sIVs and sEVs on HCEC proliferation after 24 hours of treatment. * indicates statistically significant differences between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0227] Figure 80 shows the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 48 hours. The control group was treated with PBS. * indicates statistical difference between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates the difference between sIVs or sEVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0228] Figure 81 is a line graph showing the effects of different concentration gradients of sIVs and sEVs on HCEC proliferation after 48 hours of treatment. * indicates statistically significant differences between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0229] Figure 82 shows the results of the effects of 20 μg / ml sIVs and sEVs on the proliferation of HCEC. The control group was the PBS treatment group. * indicates the statistical difference between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates the difference between sIVs or sEVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0230] Figure 83 shows the effect of 40 μg / ml sIVs and sEVs on the proliferation of HCEC. * indicates the statistical difference between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates the difference between sIVs or sEVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### P≤0.0001.

[0231] Figure 84 shows representative results of the chemotactic effect of sIVs and sEVs at different concentrations on HCEC migration after 24 hours of treatment. The control group was treated with PBS. The darker areas indicate crystal violet-stained cells; a larger number of cells indicates stronger cell migration.

[0232] Figure 85 shows the statistical results of the chemotactic effect of sIVs and sEVs at different concentration gradients on HCEC migration after 24 hours. The control group was the PBS treatment group. * indicates the statistical difference between sIVs and sEVs, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates the difference between sIVs or sEVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0233] Figure 86 shows representative results of cell scratches, showing the effects of PBS (control group), 20 μg / ml sEVs, and 20 μg / ml sIVs on HCEC migration. The control group was the PBS-treated group.

[0234] Figure 87 shows the statistical results of wound closure rate, showing the effect of PBS (control group), 20μg / ml sEVs and 20μg / ml sIVs on HCEC migration. The control group is the PBS treatment group. *Indicates the statistical difference between sIVs and sEVs, where each horizontal axis corresponds to the control group, sEVs, and sIVs from left to right in three columns. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. #Indicates the difference between sIVs or sEVs and the control group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0235] Figure 88 shows DiD-stained frozen sections of sIVs and sEVs in a mouse corneal trauma model 4 hours after exposure, reflecting the affinity of the defective corneal epithelium to MSC-sEVs and MSC-sIVs.

[0236] Figure 89 shows the images of mouse corneal fluorescein sodium staining in the 0.1 μg / μl sIVs or sEVs and PBS groups. The control group was the PBS treatment group.

[0237] Figure 90 shows the corneal epithelial defect ratios in mice treated with 0.1 μg / μl sIVs or sEVs and PBS. Lower values ​​indicate higher levels of healing. Each horizontal axis corresponds to three columns, from left to right, representing the control group, sEVs group, and sIVs group. *p<0.05, **p<0.01, ***p<0.001. The control group was treated with PBS.

[0238] Figure 91 shows corneal sensitivity threshold statistics. Larger values ​​indicate greater corneal sensitivity after recovery. Each horizontal axis corresponds to four columns, from left to right: injury-control group, injury-sEVs group, injury-sIVs group, and uninjured group. *p<0.05, **p<0.01, ***p<0.001. The control group was the PBS-treated group.

[0239] Figure 92 shows the growth of corneal neovascularization under slit lamp conditions. The alkali burn group showed thickened blood vessels and numerous neovascularization. The control group was treated with PBS.

[0240] Figure 93 shows corneal flat mounts, with the gray area indicating CD31-labeled blood vessels. The control group was the PBS-treated group.

[0241] Figure 94 shows the statistical results of corneal neovascularization, with the control group being the PBS-treated group. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. DETAILED DESCRIPTION

[0242] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0243] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

[0244] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0245] Example 1: Cell culture

[0246] 1. Extraction and culture of mesenchymal stem cells (MSCs)

[0247] Human umbilical cords were provided by Beijing Beilai Biotechnology Co., Ltd. Umbilical cords were obtained from uncomplicated normal pregnancies after cesarean section and immediately placed in saline containing penicillin (100 U / mL) and streptomycin (100 mg / mL) before being transported to the laboratory within 4 hours. After removing residual blood and blood vessels, the cords were cut into 1-3 mm pieces and digested with 0.1% type II collagenase for 1 hour at 37°C. The suspension was then filtered through a 100-mesh sieve to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco's modified Eagle's medium / Nutrient Mixture F12 complete medium (DMEM, Invitrogen, USA). The medium contained 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin (ScienCell, Carlsbad, CA). Cells were seeded in T175 culture flasks and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 3 days. When the cells reached 80% confluence, they were passaged at a 1:2 ratio, and experiments were performed using cells from P3 to P5.

[0248] 2. Culture of 293T cells

[0249] Cells were cultured using complete Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, USA) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (ScienCell, Carlsbad, CA) at 37°C and 5% carbon dioxide. The medium was changed every 2-3 days. Cells were used for various experiments at passages 3 to 6 and 70-80% confluency.

[0250] 3. Culture of HeLa cells

[0251] Cells were cultured using complete Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, USA) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (ScienCell, Carlsbad, CA) at 37°C and 5% carbon dioxide. The medium was changed every 2-3 days. Cells were used for various experiments at passages 3 to 6 and 70-80% confluency.

[0252] Example 2: Preparation of cell-derived nanovesicles

[0253] The process of producing intracellular nanovesicles is shown in Figure 1.

[0254] 1. Cell digestion and counting

[0255] When cells grow to 90% confluence, remove the supernatant and wash the cells twice with PBS. Then, trypsinize the cells and neutralize and wash the cells three times with PBS. Then, count the cells and adjust the cell number to 1×10 with PBS. 6 pieces / mL.

[0256] 2. Ultrasonic treatment of cells

[0257] Take 1×10 6 Add 2 mL of cell suspension at a density of 1 cell / mL to the bottom of a 50 mL centrifuge tube. Place an ultrasound probe in the center of the liquid. Set the ultrasound amplitude to 20% and the duration to 15 seconds, then cycle for 2 seconds on and 2 seconds off. Place the centrifuge tube on ice. Transfer the suspension to a 2 mL centrifuge tube and centrifuge.

[0258] 3. Collection of nanovesicles in small cells

[0259] Centrifugation parameters were 2000g for 10 minutes and 20,000g for 30 minutes. The supernatant was then collected and transferred to an ultracentrifuge tube and centrifuged at 150,000g for 70 minutes. All operations were performed on ice. The resulting pellet was resuspended in PBS to obtain small intracellular nanovesicles (sIVs).

[0260] 4. Collection of small extracellular vesicles

[0261] FBS is a necessary condition for culturing cells in vitro, but it contains a large number of bovine extracellular vesicles, which will also be present in complete cell culture medium containing FBS. In order to remove bovine extracellular vesicles, FBS was centrifuged at 110,000 × g at 4 ° C overnight (about 12 hours). When the cell fusion reached 60%, the cells were cultured in complete culture medium containing 10% exosome-removed FBS for 48 hours. Then, the supernatant was collected and the extracellular vesicles were separated by ultracentrifugation at 4 ° C. The specific steps include 300g × 10min, 2000g × 10min, 10,000g × 30min and 110,000g × 70min twice. The obtained precipitate was resuspended in PBS, which was small extracellular vesicles (sEVs) mainly composed of exosomes.

[0262] Example 3: Optimization of isolation parameters for intracellular nanovesicles

[0263] Taking MSCs cells as an example, referring to the steps of Example 2, the optimization process of the separation parameters of intracellular nanovesicles is shown in FIG2 .

[0264] 1. Nanoparticle size analyzer detects the difference in the yield of sIVs

[0265] PBS-resuspended sIVs were diluted to 1 ml with PBS and analyzed using Nanoparticle Tracking Analysis (NTA) software (NTA 3.3 Dev Build 3.3.104). The temperature was set to 25°C, the laser was set to Blue 488, the flow rate was set to 50, and the mode was set to automatic detection. Three injections and three analyses were performed, and the peak value average was used as the Mode particle size result. The camera mode was sCMOS, the laser type was Blue 488, and the viscosity was 0.9 cP.

[0266] 2. Transmission electron microscopy observation of the morphology of sIVs in different cells

[0267] Take the centrifuged sIVs, resuspend them in 200μL PBS solution and mix them evenly. Take 10μL of sIVs solution and mix it with 4% PFA in a volume ratio of 1:1, drop it on a clean plastic film to form a droplet, then put the front of the electron microscope carbon grid on the droplet and leave it for 20 minutes. Then negatively stain it with 10μL phosphotungstic acid for 90 seconds, bake the carbon grid dry, and observe it using a HitacW-7500 transmission electron microscope.

[0268] Figures 2A and 2B show the protein and vesicle yields of sIVs obtained according to this embodiment at an ultrasonic amplitude of 20% for exposure times of 5s, 10s, 15s, 20s, 25s, 30s, and 60s, respectively. The results show that the number of vesicles and protein yield decreased significantly when the exposure time was less than 10 seconds or greater than 20 seconds. Figures 2C and 2D show the protein and vesicle yields of sIVs obtained according to this embodiment at an ultrasonic exposure time of 15s for exposure times of 20%, 25%, 30%, 35%, and 40%, respectively. The results show that at an ultrasonic exposure time of 15s, the vesicle yield decreased significantly when the ultrasonic amplitude exceeded 25%. Figure 2E shows transmission electron micrographs of sIVs obtained according to this embodiment at an ultrasonic exposure time of 15s for exposure times of 20%, 25%, 30%, 35%, and 40%. FIG2F shows transmission electron micrographs of sIVs obtained according to this implementation procedure at an ultrasonic amplitude of 20% and ultrasonic times of 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, and 60 s.

[0269] This optimization process showed that at a 20% sonication amplitude, vesicle yield dropped sharply when the sonication duration exceeded 10 or 20 seconds. At a sonication duration of 15 seconds, vesicle yield dropped sharply when the sonication amplitude exceeded 25%. A 20% amplitude and a sonication duration of 15 seconds were the optimal parameters for collecting intracellular nanovesicles.

[0270] Example 4: sIVs have unique physical characteristics and high thermal stability

[0271] 1. Experimental instruments and materials

[0272] 1.1 Experimental Reagents

[0273] Table 1 Experimental reagents

[0274] 1.2 Experimental instruments

[0275] Table 2 Experimental instruments

[0276] 2. Experimental methods

[0277] 2.1 Transmission electron microscopy observation of the morphology of sEVs and sIVs

[0278] The centrifuged sEVs and sIVs (prepared in Example 2) were resuspended in 200 μL of PBS and mixed evenly. 10 μL of the sEVs and sIVs solution was mixed with 4% PFA in a volume ratio of 1:1 and dropped onto a clean plastic film to form a droplet. The front side of the carbon mesh was then placed on the droplet and allowed to stand for 20 minutes. The mesh was negatively stained with 10 μL of phosphotungstic acid for 90 seconds, dried, and observed using a HitacW-7500 transmission electron microscope.

[0279] 2.2. Nanoparticle size analysis to detect the particle diameter of sEVs and sIVs

[0280] PBS-resuspended sEVs and sIVs were diluted to 1 ml with PBS and analyzed using Nanoparticle Tracking Analysis (NTA) 3.3Dev Build 3.3.104 software. The temperature was set to 25°C, the laser to Blue 488, the flow rate to 50, and the mode to automatic detection. Three injections and three analyses were performed, and the peak value average was used as the particle size result. The camera mode was set to sCMOS, the laser type to Blue 488, and the viscosity to 0.9 cP.

[0281] 2.3 Analysis of protein composition of sEVs and sIVs by Coomassie Brilliant Blue staining

[0282] After BCA quantification, take an equal amount of protein sample and dilute to 20 μl with PBS. Add 5 μl of protein loading buffer (5×) and heat at 95°C for 5 minutes. Electrophoresis should be performed at 100V for 90 minutes. After electrophoresis, add Coomassie Brilliant Blue Ultrafast staining solution and incubate at room temperature for 2 hours. Rinse with pure water until clear. Photograph the gel.

[0283] 2.4 Western Blot Analysis of Exosomal Marker Protein Composition of sIVs and sEVs

[0284] After protein detection using the BCA kit, take an equal amount of each protein sample, add PBS to equal volume, add protein loading buffer (5×), and heat at 95°C for five minutes. Prepare the gel according to the SDS-PAGE kit instructions and insert the electrophoresis comb. Let it stand and stand at room temperature for 25 minutes to solidify. Place the prepared SDS gel in the pre-prepared electrophoresis tank. Remove the electrophoresis comb and inject the denatured protein into the SDS-PAGE loading tank. Add 1-4μl of marker on each side. Adjust the voltage to 60V. After the upper gel has run, increase the voltage to 100V and stop electrophoresis until the bottom bromophenol blue indicator line is 1-2cm from the bottom of the glass plate. Place the activated PVDF membrane on the SDS-PAGE surface, then place filter paper and sponge pads on both sides of the gel and membrane. Use the column to gently roll to remove bubbles in the system and clamp the electrophoresis clamp. Set the black electrode as SDS-PAGE and the red electrode as PVDF membrane, and transfer the membrane under constant voltage while placing ice packs in the electrotransfer tank to cool it down. After electrotransfer, place the PVDF membrane in a TBST solution containing 5% skim milk or 1% BSA and block it at room temperature for 2 hours. After blocking, add the primary antibody and incubate overnight on a shaker in a 4°C refrigerator. The next day, remove the primary antibody and add TBST solution to wash the PVDF membrane. Then, add the secondary antibody and incubate on a shaker at room temperature for 2 hours. After incubation, remove the secondary antibody again and add TBST solution to wash the PVDF membrane 3 times, 10 minutes each time. Finally, use ECL supersensitive luminescent liquid to develop the PVDF membrane.

[0285] 3. Statistical processing

[0286] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0287] 4. Experimental results

[0288] 4.1 Transmission electron microscopy reveals the morphology of sEVs and sIVs

[0289] sIVs were enriched from MSCs, 293T cells, and HeLa cells and examined using transmission electron microscopy. As shown in Figure 3, sEVs from all three cell types were round or horseshoe-shaped, with diameters ranging from 100 to 200 nm. sIVs were more numerous, round in shape, and less than 100 nm in diameter. Transmission electron microscopy revealed that sIVs were significantly smaller in diameter than sEVs.

[0290] 4.2 Nanoparticle tracking analysis reveals the size distribution of sEVs and sIVs

[0291] The results of nanoparticle size detection are shown in Figure 4 . The particle size distribution of sEVs from MSCs, 293T cells, and Hela cells is wide and the particle size is large, while the particle size distribution of sIVs is narrow and the particle size is small.

[0292] After statistical analysis, the results are shown in Figure 5. The average particle size of sEVs of MSC is 123.1±4.453nm, and the average particle size of sIVs is 75.28±9.067nm; the average particle size of sEVs of 293T is 121.6±6.855nm, and the average particle size of sIVs is 80.27±8.031nm; the average particle size of sEVs of Hela cells is 139.0±6.352nm, and the average particle size of sIVs is 68.53±10.04nm. In summary, the particle size of sIVs of the above three cells is smaller than that of their sEVs.

[0293] 4.3 Comparison of sEV and sIV yields at equal cell counts

[0294] To compare the yields of the two types of vesicles, we collected sEVs and sIVs from cell culture supernatants and adherent cells. 7 The number of sIVs vesicles produced by cells was 10- to 20-fold higher than that of sEVs (Figure 6A, Figure 6B, Figure 6C). 7 The protein production in cellular sIVs was 20- to 40-fold higher than that in sEVs (Figures 6D, 6E, and 6F), indicating that the production of sIVs was much higher than that of sEVs.

[0295] 4.4 Differences in protein distribution between sEVs and sIVs

[0296] Total proteins from cells, sEVs, and sIVs were separated by SDS-PAGE and stained with Coomassie Brilliant Blue to visualize the total protein distribution. The staining results showed that proteins contained in cells exhibited the most abundant bands, with multiple high-abundance protein bands; sEVs contained fewer protein types, with high-abundance proteins located around 200 kD and 70 kD; sIVs contained more protein types than sEVs, with high-abundance proteins located around 250 kD and 55 kD (Figure 7). The differences in protein distribution between different cells preliminarily suggest that sIVs and sEVs have different protein compositions and differ from the total protein distribution of both cells and sEVs. In other words, sIVs have a unique protein composition.

[0297] To further analyze the protein expression characteristics of cells, their sEVs and sIVs. Western blot was used to detect the expression of exosome marker proteins, Alix, HSP70, TSG101, CD63, and CD81 in cells, their sEVs and sIVs under equal protein conditions. The results are shown in Figure 8. The sEVs of cells expressed the most exosome marker proteins, which indicates that the sEVs obtained in this example are extracellular vesicles mainly composed of exosomes. The cells themselves also expressed a certain amount of Alix, HSP70, TSG101, and CD63; however, Alix and CD81 of sIVs were basically not expressed, and the expression levels of HSP70, TSG101 and CD63 were also much lower than those of cells and sEVs. This further shows that sIVs do not have the characteristics of exosomes, are not the precursors of exosomes in cells, nor are they fragments obtained from cell homogenates. sIVs are a unique group of intracellular vesicles.

[0298] 4.5 Comparison of the stability of sEVs and sIVs at different temperatures

[0299] To evaluate the stability of the two vesicles at different temperatures, sIVs and sEVs suspensions from three types of cells were divided into three parts and stored at different temperatures (-80°C, 4°C and 37°C). After 24 hours, the morphology, size and protein amount of sEVs and sIVs were evaluated. Both vesicles from the three cells were stable at -80°C and 4°C. However, transmission electron microscopy images showed that the morphology of sEVs was damaged at 37°C, with irregular shapes, broken vesicles and rough boundaries (Figure 9), and the number of sEVs was also reduced (Figure 10B), while the morphology and particle number of sIVs remained stable at 37°C. The above results indicate that sIVs have higher thermal stability than sEVs.

[0300] 4.6 Super-resolution imaging reveals that the distribution of sIVs in cells is different from that of sEVs

[0301] To gain a deeper understanding of the intracellular distribution of sIVs, we used proteomic analysis to identify a protein uniquely expressed by sIVs compared to sEVs, namely the IV signature protein. We then made this signature protein carry green fluorescent protein and performed intracellular imaging to visualize the morphology of sIVs in cells.

[0302] By performing proteomic analysis of sIVs and sEVs of MSCs, 293T, and Hela cells, we identified proteins that were uniquely expressed in sIVs in each cell type. By searching for the intersection of these three uniquely expressed proteins through a Venn diagram, we found a total of 106 common uniquely expressed proteins (Figure 11). Subsequently, we ranked these 106 proteins from high to low in terms of expression abundance in MSCs, 293T, and Hela cells, and displayed the top 50 proteins (Figures 12A, 12B, and 12C). We observed that the expression abundance of TMEM214 protein was the highest in MSCs, and the expression abundance of TMEM214 was also high in the other two cell types. TMEM214 is a transmembrane protein involved in cellular processes such as vesicle trafficking and protein transport (Zhao J., Xu J., Wang Y., et al. Membrane Localized GbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt. Plants (Basel). 2022 Sep 8; 11(18): 2342.). Therefore, we used green fluorescent protein (GFP) to label TMEM214 to visually visualize the intracellular status of sIVs, and used GFP-labeled CD63 as a marker for sEVs in cells.

[0303] Super-resolution microscopy combined with total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) showed that CD63 was visible on the cell membrane (Figure 13A, left), while TMEM214 was not expressed on the cell membrane (Figure 13A, left). This finding excludes the possibility that sIVs originate from cell membrane remodeling and confirms that the origin and activity site of sIVs are located inside the cell. Super-resolution microscopy using Wildfield-2DSM scanning mode provides an overview of protein expression throughout the cell, and images show that both CD63 and TMEM214 are significantly expressed inside the cell (Figure 13B). Subsequently, under wide-field conditions, we observed the dynamic changes in the structures of these protein markers in living cells. Pictures were taken every 10 seconds for 15 minutes to form a dynamic video. In the video screenshot, the dynamic release of sEVs from the cell membrane to the extracellular space can be observed (Figure 13C, left, white arrow), while TMEM214-labeled sIVs are diffusely distributed within the cell and are not released extracellularly (Figure 13C, right). Intuitive microscopic imaging shows that sIVs are diffusely distributed in the cell in a cloud-like manner and are not released extracellularly.

[0304] 5. Summary

[0305] In this example, we used MSCs, 293T and Hela cells as examples to collect and characterize sIVs and sEVs. Transmission electron microscopy and nanoparticle size analysis revealed that sIVs were significantly smaller than sEVs; the total protein expression patterns of cells, sEVs and sIVs were different, with sIVs expressing low exosome marker proteins; and at the same cell number, the yield of sIVs was significantly greater than that of sEVs. At -80°C, the stability of sIVs and sEVs was comparable, but at 37°C, the stability of sIVs was significantly better than that of sEVs. Super-resolution imaging observed that sEVs were released from the cell membrane to the outside of the cell, while sIVs were active inside the cell. In general, different cells can be collected by the method described in the present invention to obtain a group of sIVs vesicles with the same properties, which contain a unique protein composition, are highly stable at physiological temperature, and have a yield much higher than that of extracellular vesicles.

[0306] Example 5: Quantitative proteomic analysis shows that sIVs have unique protein expression profiles

[0307] 1. Experimental instruments and materials

[0308] 1.1 Experimental Reagents

[0309] Table 3 Experimental reagents

[0310] 1.2 Experimental Instruments

[0311] Table 4 Experimental instruments

[0312] 2. Experimental methods

[0313] 2.1 Sample preparation for protein profiling of cells and their sEVs and sLVs

[0314] 2.1.1 Protein extraction

[0315] 1) Add 220 μl of urea lysis buffer (8 M urea, 50 mM NH 4 HCO 3 , protease inhibitors) to each of the cells, sEVs, and sIVs (prepared in Example 2) and lyse at room temperature for 5 minutes.

[0316] 2) Ultrasonic disruption on ice (energy 35%, ON 3s, OFF 3s, total ultrasonic time 2 min), insert the sample tube into an ice box, set the centrifuge temperature to 20°C, centrifuge at 14,000g for 10 min, remove the supernatant, and repeat the centrifugation once.

[0317] 3) Protein concentration was determined by BCA assay, and 100 μg protein was collected from each of cells, sEVs, and sIVs.

[0318] 2.1.2 Protein denaturation

[0319] 1) Add DTT to each sample tube of cells, sEVs, and sIVs to a final concentration of 10 mM and incubate at 37°C for 1 hour to reduce the proteins.

[0320] 2) Add IAA to each sample tube containing cells, sEVs, and sIVs to a final concentration of 40 mM and incubate at room temperature for 1 hour in the dark.

[0321] 3) First, label the sample number on the collection tube. Equilibrate a 10 kDa ultrafiltration tube twice with 150 μl of HPLC-grade methanol each time. Centrifuge at 14,000 g for 5 min, then add 300 μl of 50 mM NH4HCO3. Rinse twice, add 100 μg of the reductively alkylated protein sample, centrifuge at 14,000 g for 20 min at 4°C, and rinse three times with 300 μl of 50 mM NH4HCO3. Replace the collection tube with a new one, and add 75 μl of 50 mM NH4HCO3 to the ultrafiltration tube.

[0322] 2.1.3 Protease cleavage

[0323] 1) Add 3 μg of trypsin for mass spectrometry and incubate in a 37°C incubator for 14-16 hours.

[0324] 2) The next day, centrifuge at 14,000 g for 20 minutes at 4°C, add 50 μl of 50 mM NH4HCO3, rinse twice, add 1% (volume ratio) formic acid to the collection tube to terminate the enzyme digestion, and evaporate to dryness in vacuo at 60°C.

[0325] 2.1.4 Library construction and fractionation

[0326] 1) Resuspend the samples in 30 μl of 0.1% formic acid and measure the concentration using a nanodrop. Next, remove approximately 10 μg of peptides from each sample and combine them into one sample, S.

[0327] 2) 6 μg of sample S was taken out for mass spectrometry analysis in DDA mode, and the remaining S sample was fractionated using a homemade high pH reverse phase column.

[0328] 3) For fractionation, prepare the required reagents according to Table 5. Buffer A is 100% acetonitrile (ACN), and Buffer B is 0.1% trifluoroacetic acid (TFA).

[0329] Table 5 Fractionation reagent ratio

[0330] 4) Use a wire to load a C18 membrane into a 200 μl pipette tip. Next, dissolve 30 mg of high-pH-resistant C18 filler in 200 μl of Buffer A and add the filler to the fractionating column. Centrifuge at 3,000 g for 2 minutes at 4°C. Wash once with 200 μl of Buffer A and three times with 200 μl of Buffer B. Finally, set the column aside for future use.

[0331] 5) Load the cleaved peptides onto the fractionation column and repeat the loading process 5 times.

[0332] 6) Wash the column three times with 200 μl of Buffer B. Next, add 150 μl of elution buffer of varying concentrations and perform gradient elution. Combine the 6% and 35% fractions into one fraction, and extract 1.5 μg of peptides from each fraction for DDA mass spectrometry analysis.

[0333] 2.2 Liquid chromatography mass spectrometry parameters of cells and their sEVs and sIVs

[0334] The enzymatically digested peptides were loaded onto a homemade Trap column (100 μm × 2 cm, C18 filler, 3 μm particle size, 120A) using phase A (containing 0.1% formic acid, 2% acetonitrile, and 97.9% water) at a flow rate of 3 μl / min. Subsequently, the Trap column was eluted using different gradients of phase B (containing 97.9% acetonitrile, 2% water, and 0.1% formic acid). These eluted peptides passed through an analytical column (150 μm × 15 cm, C18 filler, 1.9 μm particle size, 120A) to form an electrospray and ultimately entered the mass spectrometer detector.

[0335] The gradient of phase B was set as follows: 0 min to 5%, 2 min to 10%, 65 min to 22%, 91 min to 35%, 92 min to 80%, 105 min to 80%, 106 min to 5%, and 120 min to 5%. The flow rate was maintained at 500 nL / min throughout the process.

[0336] For DDA scans, the mass spectrometer parameters were set as follows: a TOF MS accumulation time of 0.25 seconds, a mass scan range of 300–1500 Daltons (Da), detecting only ions with charges of +2 to +5, and a mass deviation of less than 50 ppm. A maximum of 60 ions were monitored per cycle, and after each detection, the detected ions were isolated for 16 seconds. Dynamic fragmentation mode was used for fragmentation energy. The accumulation time for product ions was 0.04 seconds, and high-sensitivity scanning mode was used.

[0337] During DIA scanning, the mass spectrometer parameters differed: the TOF MS had an accumulation time of 0.05 seconds, and the secondary scan was performed in high-sensitivity mode. The number of variable windows was set to 100, with an accumulation time of 30 milliseconds per window. The mass scan range was also 300–1500 Da. The specific mass range for each variable window was calculated using the SWATH Variable Window Calculator_V1.1 program.

[0338] 2.3 Cell and sEVs / sIVs protein profile data processing and bioinformatics analysis

[0339] Proteinpilot software (version 5.0.1) was used to perform database searches on the raw data collected using the DDA mode, using trypsin as the digestion method. The Uniprot human database, which contains 20,431 annotated proteins and was released in July 2019, was used. The screening criteria were an unused protScore greater than 0.05. The Proteinpilot search results were imported into SWATH software (version 2.0) as a database for quantification of the DIA mode data.

[0340] During quantification, six peptides were selected for each protein, and six transitions (ion pairs) were selected for each peptide. Peptide confidence was set to 99%, and the FDR (false positive rate) was set to 1%. Modified peptides were excluded, the peak extraction window was set to 10 minutes, and the mass deviation was controlled within 50 ppm. Two endogenous peptides were selected every 10 minutes to correct for retention time, and the output peak area was used as the quantitative value.

[0341] Processing of protein expression data involved the following steps: First, raw quantitative values ​​were log2 transformed to conform to a normal distribution and then normalized using the normalize.quantiles function in the preprocessCore package in R. After removing proteins without gene names, differential expression analysis was performed using the stats package in R. Proteins with a P-value less than 0.05 and a fold change greater than 1.5 were selected as differentially expressed proteins. Corrected p-values ​​were set to less than 0.05.

[0342] GO and pathway analyses were performed using the Cytoscape plugin clueGo. For GO enrichment analysis, cellular component (CC), molecular function (MF), and biological process (BP) were selected. For pathway enrichment analysis, the kegg and reactome databases were used.

[0343] Heatmaps, principal component analysis (PCA) scores, Venn diagrams, and volcano plots were performed using R or Hiplot software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Metascape online analysis software.

[0344] 2.4 Enzyme-linked immunosorbent assay

[0345] MSC-derived sEVs and sIVs were collected, adjusted to equal mass, and performed according to the instructions for the enzyme-linked immunosorbent assay (ELISA) kit. The samples were added to the antibody-coated wells and incubated for 2 hours, and the plate was washed. Biotin was added, incubated for 1 hour, and the plate was washed. HRP was added, incubated for 1 hour, and the plate was washed. TMB substrate was added and incubated for 20 minutes. STOP solution was added until color was apparent. The absorbance of each well in the plate was read using a microplate reader at wavelengths of 450 nm and 540 nm.

[0346] 3. Statistical processing

[0347] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0, and statistical analysis between the two groups was performed using the t test. P values ​​< 0.05 were considered statistically significant.

[0348] 4. Experimental results

[0349] 4.1 Venn diagram showing differences in protein composition between cells and their sEVs and sIVs

[0350] To characterize the molecular composition of sEVs and sIVs, we used label-free mass spectrometry to perform proteomic analysis of sEVs and sIVs derived from MSCs, 293T cells, and HeLa cells, and compared the results with the cells. 2744, 3018, and 2412 proteins were identified in MSCs, 293T cells, and HeLa cells, respectively. Meanwhile, 1678, 2310, and 1427 proteins were detected in sEVs from these three cell types, respectively, and 2066, 2403, and 2272 proteins were found in sIVs from these three cell types (Figure 14). There was some overlap in the protein types between them, but they were not identical. In all three cell types, the protein content in sIVs was more diverse than that in sEVs.

[0351] 4.2 Principal component analysis reveals differences in protein composition of cells and their sEVs and sIVs

[0352] PCA analysis of the protein components of cells, sEVs, and sIVs (Figure 15) revealed distinct protein distribution patterns among cells, sEVs, and sIVs. sEVs and sIVs exhibited significant differences in protein expression across the three different cell types, suggesting that sIVs possess distinct protein expression profiles distinct from sEVs.

[0353] 4.3 Quantifiable protein differential analysis of cells and their sEVs and sIVs

[0354] Further statistics showed that there were 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, of which 753 were significantly downregulated in sIVs compared with sEVs, 672 were significantly upregulated, 468 of which had a difference of more than 10 times, 227 were significantly downregulated in sIVs compared with sEVs, and 241 were significantly upregulated (Figure 16A). There were 1841 differentially expressed proteins in 293T cells, of which 780 were significantly downregulated in sIVs compared with sEVs, and 10 were significantly upregulated. There were 1061 differentially expressed proteins in Hela cells, of which 321 had a fold difference greater than 10-fold, 159 were significantly downregulated in sIVs compared with sEVs, and 162 were significantly upregulated (Figure 16B). There were 1474 differentially expressed proteins in Hela cells, of which 594 were significantly downregulated in sIVs compared with sEVs, and 880 were significantly upregulated, of which 341 had a fold difference greater than 10-fold, 143 were significantly downregulated in sIVs compared with sEVs, and 198 were significantly upregulated (Figure 16C). This further demonstrates the uniqueness of sIVs.

[0355] The most significantly upregulated and downregulated proteins in sEVs and sIVs were not identical across the three cell types (Figure 17). However, membrane-associated proteins were expressed at lower levels in sIVs, while endoplasmic reticulum and ribosome-associated proteins were more abundant in sIVs. This initially suggests that sIVs are distinct entities produced by cells, rather than precursors or fragments of cell lysates or extracellular vesicles.

[0356] 4.4 Differences in the expression of exosomal markers in cells and their sEVs and sIVs

[0357] We extracted the list of exosome markers recommended by MISEV2018 (Théry C., Witwer KW, Aikawa E., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines [J]. Journal of extracellular vesicles, 2018, 7(1): 1535750.) and compared sEVs and sIVs from the three cell types with these markers. sEVs showed higher expression levels of exosome markers, while sIVs had lower expression levels of most exosome marker proteins (Figure 18). This further indicates that sIVs do not have exosome characteristics and are unique vesicles from the inside of cells.

[0358] 4.5 Differential expression of organelle marker proteins in cells and their sEVs and sIVs

[0359] We also compared the organelle protein expression profiles of sEVs and sIVs in the three cell types. Overall, sIVs contained higher levels of intracellular organelle proteins than sEVs (Figure 19). In particular, sIVs showed elevated expression levels of proteins associated with membrane-enriched organelles such as endosomes, the endoplasmic reticulum, and the Golgi apparatus. In contrast, sEVs contained more abundant cellular membrane proteins (Figure 19). This suggests that sIVs possess intracellular characteristics.

[0360] 4.6 Differential expression of the Clathrin protein family in cells and their sEVs and sIVs

[0361] Among the proteins contained in intracellular vesicles, the Clathrin family of proteins is crucial for vesicle organization and activity. Clathrin proteins play a key role in intracellular transport by promoting cargo transport between organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways. Given the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that the expression of most clathrin family proteins was upregulated in sIVs, while sEVs showed low expression (Figure 20). This finding suggests that the sIVs we isolated may participate in communication between different cellular compartments within the cell.

[0362] 4.7 Enrichment analysis of sIVs-specific proteins

[0363] In Example 4, we analyzed 106 proteins expressed by three cellular sIVs that were distinct from sEVs. These proteins are unique to sIVs and can represent the characteristics of sIVs. We performed gene enrichment analysis on these proteins. In terms of cellular components (CC), these proteins were associated with COPII-coated ER-Golgi transport vesicles, transport vesicles, coated vesicles, ER-Golgi transport vesicle membranes, ER-Golgi intermediate compartments, transport vesicle membranes, and coated vesicle membranes (Figure 21). Furthermore, in the biological process (BP) category, terms such as glycerophospholipid biosynthesis process, response to ER stress, ubiquitin-dependent ERAD pathway, intracellular protein transport, and ER-Golgi vesicle-mediated transport were found (Figure 22). This result indicates that the sIVs we isolated are intrinsic vesicle components within cells.

[0364] 4.8 Comparison of cytokines contained in sEVs and sIVs

[0365] Intracellular vesicles are involved in the intracellular transport of various secretory factors, while exosomes carry these factors to the extracellular space. Therefore, we compared the levels of cytokines carried by sEVs and sIVs. Proteomic analysis showed that sIVs had lower levels of interleukin-1β (IL-1β) and insulin-like growth factor 2 (IGF-2) compared to sEVs (Figure 23). Subsequently, we used ELISA to further quantify low-abundance cytokines. The results showed that, based on the same mass, sIVs contained high levels of insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and interleukin-10 (IL-10) (Figure 24A, Figure 24B, Figure 24C). Meanwhile, there was no significant difference in IL-6 levels between sEVs and sIVs ( FIG. 24D ), and the level of Tumor Necrosis Factor α (TNFα) ( FIG. 24E ) was lower in sIVs.

[0366] 5. Summary

[0367] In this example, we used proteomics to characterize the protein composition of cells, sEVs, and sIVs using MSCs, 293T cells, and HeLa cells as examples. The results showed that sIVs had a unique protein expression profile, distinct from cells and sEVs. sIVs expressed low levels of exosome markers and high levels of markers for membrane-enriched organelles and the Clathrin family of proteins within the cell, suggesting that sIVs play a role in intracellular transport and mediate intracellular organelle communication. Gene enrichment analysis of sIVs directly implicated sIVs in transport between the endoplasmic reticulum and the Golgi apparatus, including vesicle-mediated forward transport from the endoplasmic reticulum to the Golgi apparatus and retrograde vesicle-mediated transport from the Golgi apparatus back to the endoplasm. These findings strongly suggest that sIVs play a key role in intracellular transport, particularly in mediating inter-organelle communication. Among these, COP-coated vesicles have been widely reported to participate in intracellular transport initiated by the endoplasmic reticulum. During this process, correctly folded and assembled proteins in the endoplasmic reticulum (ER) are encapsulated into COP-coated transport vesicles, which then detach from the ER membrane. Subsequently, the vesicles shed their coating and fuse with each other to form tubular clusters of vesicles. The Golgi apparatus is responsible for modifying the proteins and lipids received from the ER and distributing them to the cell membrane, endosomes, and secretory vesicles. Within the Golgi apparatus, proteins and lipids move in a cis-to-trans direction, completing this process through vesicular transport. Proteomic analysis further confirmed that sIVs are involved in intracellular vesicular transport and are closely associated with the ER, Golgi apparatus, and COP-coated vesicles.

[0368] The above results indicate that sIVs are completely different from sEVs, sIVs play an important role in intracellular substance transport, and sIVs are a unique group of vesicles.

[0369] Example 6: sIVs have unique miRNA expression profiles

[0370] 1. Experimental instruments and materials

[0371] 1.1 Experimental Reagents

[0372] PBS, purchased from Gibco Biotechnology (USA)

[0373] 1.2 Experimental instruments

[0374] Table 6 Experimental instruments

[0375] 2. Experimental methods

[0376] 2.1 Data Collection and Analysis

[0377] 2.1.1 RNA isolation, library preparation, and sequencing

[0378] After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS and tested for RNA degradation and contamination on a 1% agarose gel. Check RNA purity using a spectrophotometer. RNA concentration was determined using Qubit in Flurometer TM RNA was measured using the RNA Nano 6000 assay kit on the Agilent Bioanalyzer 2100 system.

[0379] 2.1.2 Library Preparation for Small RNA Sequencing

[0380] 3 μg of total RNA from each sample was used as the input sample for the small RNA library. Small RNA Library Prep Set for Generate and add index codes to assign sequences to each sample. Amplification was performed on a PCR instrument using LongAmp Taq 2X Master Mix, SR Primer for illumina, and index (X) primers. PCR products were then purified on an 8% polyacrylamide gel (100V, 80 minutes). DNA fragments corresponding to 140 to 160 bp were recovered and dissolved in 8 μL elution buffer. Finally, library quality was assessed on an Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.

[0381] 2.1.3 Cluster Generation and Sequencing

[0382] The indexed samples were clustered using the TruSeq SR Cluster Kit v3-cBot-HS (Illumia) on the cBot Cluster Generation System according to the manufacturer's instructions. After cluster generation, sequencing was performed on the Illumina HiSeq 2500 / 2000 platform to generate 50 bp single-end reads for library preparation.

[0383] 2.1.4 Data Analysis

[0384] 1) Quality Control

[0385] First, the raw data (raw reads) in fastq format were processed by custom Perl and Python scripts. In this step, clean reads were obtained from the raw data by removing reads containing ploy-N, 5' end joint contamination, no 3' end joint or insertion tag, containing ploy A or T or G or C, and low quality. At the same time, the Q20, Q30 and GC content of the raw data were calculated. Then, a certain length range was selected from the clean reads for all downstream analyses. Bowtie (Langmead B., Trapnell C., Pop M., Salzberg SLU ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genome biology, 2009, 10 (3): R25.) was used to map the small RNA tags to the reference sequence, without allowing mismatches, to analyze their expression and distribution on the reference sequence.

[0386] 2) Alignment of known miRNAs

[0387] The mapped small RNA signatures were used to identify known miRNAs. Using miRBase 20.0 as a reference, modified versions of the software mirdeep2 and srna-tools-cli were used to identify potential miRNAs and map their secondary structures. Custom scripts were used to obtain the miRNA count and base bias at the first position of identified miRNAs of a specific length, as well as the miRNA count and base bias at each position for all identified miRNAs.

[0388] 3) Summary of small RNA annotation

[0389] Summarize all previously obtained alignments and annotations. In the previous alignments and annotations, some small RNA tags may be mapped to multiple categories. To ensure that each unique small RNA is mapped to only one annotation, we follow the following priority rule: known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>new miRNA.

[0390] 4) Data Analysis

[0391] Target gene prediction was performed using miRanda, and miRNA target gene prediction was performed using the intersection of miRanda and RNAhybrid software. Differentially expressed miRNA input data were read count data obtained from miRNA expression level analysis. For samples with biological reproducibility: differential expression analysis between two conditions / groups was performed using the DESeq R package (3.0.3). P values ​​were adjusted using the Benjamini & Hochberg method. By default, the adjusted P value was set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, Venn diagrams, volcano plots, etc. were drawn online using Hiplot and adjusted using Adobe Illustrator.

[0392] 5) GO and KEGG enrichment analysis

[0393] GO enrichment analysis was performed on candidate target genes of differentially expressed miRNAs (hereafter referred to as "target gene candidates"). GO enrichment analysis was performed using GOseq, which is based on the Wallenius noncentered hypergeometric distribution and adjusts for gene length bias. KOBAS software was used to test the statistical enrichment of target gene candidates in Kyoto Encyclopedia of Genes and Genomes pathways.

[0394] 3. Experimental results

[0395] Relative RNA abundance in sEVs and sIVs of MSCs, 293T, and HeLa cells

[0396] First, we used a bioanalyzer to analyze the RNA abundance of sEVs and sIVs in MSCs, 293T cells, and HeLa cells (Figure 25). The results showed that there was no statistical difference in the RNA abundance of sEVs and sIVs among the three cell types, with MSCs showing higher RNA abundance than the other two cell types, which may be related to the rich biological effects of MSCs.

[0397] 3.2 Distribution of small RNAs in sEVs and sIVs

[0398] Small RNAs associated with cells and extracellular vesicles have been a hot topic of research in recent years. MicroRNAs (miRNAs), in particular, possess diverse biological functions and serve as biomarkers for a variety of diseases. We analyzed the small RNA species in sEVs and sIVs. The alignment and annotation of each small RNA species with total RNA were summarized. Because a single sRNA can be mapped to several different annotations simultaneously, to ensure a unique annotation for each unique sRNA, we prioritized small RNAs according to the order of known miRNAs > rRNAs > tRNAs > snRNAs > snoRNAs > YRNAs > repeats > genes > novel miRNAs, and calculated the proportion of each small RNA in total RNA. Results showed that YRNAs were the predominant RNA in sEVs, while miRNAs were the predominant RNA in sIVs. MiRNAs accounted for 29.15% of MSC-derived sEVs and 92.52% of sIVs; miRNAs accounted for 13.29% of 293T cell-derived sEVs and 59.42% of sIVs; and miRNAs accounted for 32.22% of HeLa cell-derived sEVs and 58.09% of sIVs. The proportion of miRNAs in sIVs of all three cell types was significantly higher than in sEVs (Figure 26). Among the three cell types, MSC-derived sIVs had a more pronounced miRNA profile, further suggesting that MSC-derived vesicles, similar to MSCs, may possess a rich set of biological regulatory functions.

[0399] 3.3 Global miRNA expression characteristics of sEVs and sIVs

[0400] MiRNAs have a rich array of biological regulatory roles and comprise a significant proportion of small RNAs. Therefore, we conducted a subsequent analysis of miRNAs, using a Venn diagram to analyze the miRNA species contained in sEVs and sIVs of the three cell types. The results showed that 694, 803, and 630 miRNAs were detected in sEVs of MSCs, 293T cells, and HeLa cells, respectively, while 989, 902, and 795 miRNAs were found in sIVs of these three cell types (Figure 27). There was some overlap in the miRNA species among these three cell types, but they were not identical. MSCs contained the most abundant miRNAs, while sEVs and sIVs contained a higher abundance of miRNAs.

[0401] Because sEVs and sIVs contain commonly expressed miRNAs, principal component analysis was subsequently used to compare the miRNA expression patterns of sEVs and sIVs. The results showed that the miRNA components contained in the two vesicles were quite different and the expression patterns were unrelated (Figure 28), further demonstrating that sIVs are different from sEVs and contain a unique miRNA expression profile.

[0402] 3.4 Highly Abundant miRNAs in sEVs and sIVs

[0403] Analysis of the top 10 most abundant miRNAs in sEVs and sIVs revealed that miR-148a-3p and let-7i-5p were highly expressed in sEVs from all three cell types, while let-7f-5p was highly expressed in sIVs from all three cell types. Furthermore, miRNAs such as miR-148-3p, miR-21-5p, and miR-100-5p were highly expressed in sEVs and sIVs from multiple cell types (Figure 29).

[0404] 3.5 Analysis of differentially expressed miRNAs in sEVs and sIVs

[0405] To further compare the differences in miRNA expression between sEVs and sIVs, we performed differential expression analysis on sEVs and sIVs of the three cell types. The results showed that in MSCs, sEVs and sIVs had 70 differential miRNAs, of which 22 were significantly downregulated and 48 were significantly upregulated in sIVs compared with sEVs; in 293T cells, sEVs and sIVs had 167 differential miRNAs, of which 125 were significantly downregulated and 42 were significantly upregulated in sIVs compared with sEVs; and in Hela cells, sEVs and sIVs had 120 differential miRNAs, of which 55 were significantly downregulated and 65 were significantly upregulated in sIVs compared with sEVs (Figures 30-31). This further illustrates that sIVs and sEVs are different.

[0406] 3.6 Enrichment analysis of differentially expressed miRNA target genes in sEVs and sIVs

[0407] MiRNAs exert their biological effects by regulating downstream target genes. Therefore, after comparing the differentially expressed miRNAs in each group, we performed gene enrichment analysis on the target gene sets of these miRNAs, including GO analysis and KEGG analysis. For the sake of convenience, we will refer to "target genes of differentially expressed miRNAs" as "candidate target genes." The results of GO enrichment analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to intracellular metabolic processes, their cellular localization was intracellular membrane-related organelles, and their molecular functions were related to protein binding and enzyme metabolic reactions (Figure 32A). KEGG pathway analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to pathways such as axon guidance, cell differentiation, endocytosis, and immune regulation (T cell receptor signaling pathway, B cell receptor signaling pathway) (Figure 32B). GO enrichment analysis results of 293T cells showed that candidate target genes of sEVs and sIVs were also related to intracellular processes, with cellular localization being the cell membrane, endomembrane system, and organelle membrane, and molecular functions being related to protein binding and enzyme metabolic reactions (Figure 33A). KEGG pathway analysis showed that candidate target genes of sEVs and sIVs in 293T cells were associated with multiple signaling pathways such as CAMP and Ras (Figure 33B). GO enrichment analysis results of Hela cells showed that candidate target genes of sEVs and sIVs were also related to intracellular processes, with cellular localization being the cell membrane, endomembrane system, and organelle membrane, and molecular functions being related to protein binding and enzyme metabolic reactions (Figure 34A). KEGG pathway analysis showed that candidate target genes were associated with multiple signaling pathways such as synapses and MAPK (Figure 34B).

[0408] 4. Summary

[0409] In this example, we used small RNA sequencing technology to analyze the small RNA composition of sEVs and sIVs using MSCs, 293T, and HeLa cells, and further explored the expression patterns of miRNAs. Intracellular miRNAs are produced in the nucleus and transported to the cytoplasm, where they participate in the regulation of target genes. Therefore, miRNAs produced intracellularly must primarily perform their biological functions by regulating gene expression and participating in various cellular biological processes, such as cell proliferation, differentiation, and apoptosis. Many miRNAs have been found to be specifically expressed in different stem cell types, regulating cell differentiation and the maturation of specific cell lineages. Other miRNAs may promote or inhibit cell death signaling pathways, thereby affecting cell survival and apoptosis. They can maintain cellular homeostasis by regulating apoptosis-related genes, such as BCL2 family members, caspases, and p53. Within cells, miRNAs can act as signaling regulators to regulate cellular biological processes, such as growth factor signaling, responses to oxidative stress, and inflammatory responses. They can target key molecules in specific signaling pathways, thereby affecting the entire signaling pathway. Our experimental results indicate that sIVs are rich in miRNAs, suggesting that they may play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis, and signaling pathways, and thus possess significant potential for application. Our results revealed that sIVs exhibited a unique miRNA expression profile, significantly different from that of sEVs, with a higher abundance of miRNAs in sIVs. This finding suggests that sIVs have potential biological regulatory roles and may facilitate communication between different intracellular organelles. Candidate gene enrichment analysis of differentially expressed miRNAs in sEVs and sIVs further revealed that sIVs are closely associated with intracellular membrane-like organelles. In summary, sEVs and sIVs differ in their small RNA composition, particularly miRNAs. This miRNA enrichment analysis further confirms that sIVs play an important role in intracellular cargo transport. Compared to sEVs, sIVs contain a more diverse miRNA population and may possess a wider range of biological functions. These findings provide important insights into the functions of sIVs in cell biology.

[0410] Example 7: sIVs have unique lipidomic characteristics

[0411] 1. Experimental instruments and materials

[0412] 1.1 Experimental Reagents

[0413] Table 7 Experimental reagents

[0414] 1.2 Experimental instruments

[0415] Table 8 Experimental instruments

[0416] 2. Experimental methods

[0417] 2.1 Metabolite extraction

[0418] After separation and enrichment of sEVs and sIVs (prepared in Example 2), the samples were resuspended in PBS. 200 μL of water was added to the container, followed by 480 μL of an extract, which was a mixture of MTBE and MeOH in a ratio of 5:1 and contained an internal standard substance. The mixed solution was quickly placed in a liquid nitrogen tank and frozen for 1 minute, then taken out to thaw and mixed by a vortex mixer for 30 seconds to make the solution uniform. The above freezing, thawing and mixing steps were repeated 3 times, followed by ultrasonic treatment in an ice-water bath for 10 minutes. The treated sample was allowed to stand at -40°C for 1 hour. Then, the sample was separated by centrifugation at 3000 rpm (centrifugal force 900 × g, radius 8.6 cm) for 15 minutes at 4°C. 300 μL was taken out from the supernatant, transferred to an EP tube, and vacuum dried. 100 μL of a complex solution (DCM:MeOH=1:1) was added to the dried sample, vortexed for 30 seconds, and ultrasonically treated again in an ice-water bath for 10 minutes. Finally, centrifuge at 13,000 rpm (centrifugal force 16,200 × g, radius 8.6 cm) for 15 min at 4°C, and transfer 75 μL of the supernatant to a sample injection bottle for testing on an instrument.

[0419] 2.2 Metabolite Detection

[0420] The target compounds were separated chromatographically using a Vanquish ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column. Phase A consisted of 10 mmol / L ammonium formate in 40% water and 60% acetonitrile; phase B consisted of 50 mL / 1000 mL (10 mmol / L) ammonium formate in 10% acetonitrile and 90% isopropanol. The following gradient elution program was used: 40% B from 0 to 1.0 min; linear increase to 100% B from 1.0 to 12.0 min; hold at 100% B from 12.0 to 13.5 min; linear decrease to 40% B from 13.5 to 13.7 min; and hold at 40% B from 13.7 to 18.0 min. The mobile phase flow rate was set at 0.3 mL / min, the column temperature was 55 °C, the sample tray temperature was 4 °C, and the injection volume was 2 μL (positive and negative ion modes).

[0421] Primary and secondary mass spectrometric data were collected simultaneously using a Thermo Q Exactive HFX mass spectrometer under the control of Xcalibur control software (version 4.0.27, Thermo). Specific parameters were as follows: sheath gas flow rate of 10 Arb, capillary temperature of 350°C, full MS resolution of 120,000, MS / MS resolution of 7500, collision energy of 10 / 30 / 60 in NCE mode, and spray voltage of 4 kV (positive ion mode) or -3.8 kV (negative ion mode).

[0422] 2.3 Data Analysis

[0423] ProteoWizard software was used to convert mass spectra into mzXML format. Retention time correction, peak identification, peak extraction, peak integration, and peak alignment were then performed using XCMS, with a minfrac value of 0.5 and a cutoff value of 0.3. Lipid identification was performed using XCMS, a custom R package, and the lipidblast database. Bioinformatics plots were created online using Hiplot and adjusted using Adobe Illustrator.

[0424] 3. Experimental results

[0425] The ionization source of the Orbitrap platform is electrospray ionization, which has two ionization modes: positive ion mode (POS) and negative ion mode (NEG). Combining the two modes when detecting metabolites can achieve higher metabolite coverage and better detection effects. During data analysis, one ion mode is generally selected. This study uses the positive ion mode as an example for analysis.

[0426] 3.1 Proportions of various metabolites in sEVs and sIVs of MSCs, 293T, and HeLa cells

[0427] Secondary spectra in lipidomics are incidental, so only lipid species identified across all groups within a set of comparisons are reliable. Therefore, we categorized and statistically analyzed the metabolites identified by different cell types based on their chemical classification information. The proportions of each metabolite are shown in Figure 35. 31 metabolites were identified in MSCs, 43 in 293T cells, and 44 in HeLa cells. Differential expression levels of various lipid species were observed in sEVs and sIVs. PC and PE are common lipid components of cell membranes. The results in Figure 35 show that the proportions of PE and PC in vesicles from all three cell types were high, indicating the presence of abundant biomembrane structures in sEVs and sIVs.

[0428] 3.2 Total lipid expression characteristics in sEVs and sIVs of MSCs, 293T, and HeLa cells

[0429] Metabolomics data are high-throughput, and principal component analysis can effectively highlight the overall distribution trends of metabolomics data and the degree of differences between sample groups. The results showed that sEVs and sIVs from MSCs, 293T cells, and HeLa cells had different lipid distribution patterns (Figure 36), indicating that sIVs are a unique vesicle population, distinct from sEVs, with significantly different lipid expression patterns.

[0430] 3.3 Differential lipid expression characteristics in sEVs and sIVs of MSCs, 293T cells, and HeLa cells

[0431] Heat maps can visually display the overall distribution of metabolite differences between groups. We visualized the results of the differential metabolite screening in the form of heat maps. The results of the sIVs group versus the sEVs group are shown in Figure 37. Non-cancerous cell sIVs highly express common biomembrane structural lipids such as PC, PI, and PE. However, the lipid expression pattern of HeLa cell sIVs differs from that of non-cancerous cells, which may be related to abnormal lipid metabolism in cancer cells.

[0432] 3.4 Differential lipid content changes and classification information in sEVs and sIVs of MSCs, 293T cells, and Hela cells

[0433] The lipidome histogram visualizes the changes in metabolite content and their classification information. The results for the sIVs group versus the sEVs group are shown in Figure 38. Each bar in the lipidome histogram represents a metabolite. In MSCs, PC, PI, PE, PG, and OxPI were significantly overexpressed in sIVs, with PC over 200-fold higher in sIVs. In 293T cells, PE, Cer / AS, Cer / AP, Cer / ADS, PI, and PC were significantly overexpressed in sIVs, with PE over 900-fold higher in sIVs. In Hela cells, SHexCer, CE, PI, and HexCer / NS were significantly overexpressed in sIVs, with SHexCer over 400-fold higher in sIVs.

[0434] 4. Summary

[0435] Lipidomics identifies and quantifies various lipid molecules. Lipids are classified into eight major categories: fatty acyl groups, glycerolipids, phospholipids, sterol lipids, allyl lipids, sphingolipids, glycolipids, and polyketides. Cell membranes primarily contain various phospholipids, which can be further divided into glycerophospholipids and sphingomyelins, which have distinct differences. Glycerophospholipids are primarily located in the inner leaflet of the phospholipid bilayer of the cell membrane and, along with cholesterol, constitute the primary component of the cell membrane. In this example, sIVs contain a high concentration of glycerophospholipids, such as PC and PE. Sphingomyelins are a class of phospholipids containing a sphingosine group. Sphingomyelins are located in the outer leaflet of the cell membrane and are primarily involved in neuronal activity and signaling. In this example, sEVs contain a high concentration of sphingomyelins, such as SM. Furthermore, glycerophospholipids are involved in many other physiological processes in the body, such as energy metabolism and hormone synthesis; sphingomyelins play a relatively minor role in these processes. Overall, sEVs and sIVs from MSCs, 293T cells, and HeLa cells exhibit distinct lipid distribution patterns. The expression levels of PC and PE are high in sIVs, with PC being more than 200 times more expressed in sIVs. PC, also known as lecithin, is known as the "third nutrient" alongside proteins and vitamins, and plays many important biological roles. Lecithin can increase the axonal growth of neurons, promote brain development, enhance memory, and prevent Alzheimer's disease. In addition, PE is one of the main molecules that make up the skeleton of biological membranes. Its unique structure, including a phosphate group, a glycerol group, an acyl group, and an ethanolamine, enables it to form stable non-lamellar and multilamellar liposome vesicles in biological membranes. This structure provides a stable foundation for biological membranes and helps maintain the normal structure and function of cells.

[0436] Interestingly, PC and PE, which are highly expressed in sIVs, are both glycerophospholipids. Since the endoplasmic reticulum is the site of glycerophospholipid synthesis, it is reasonable that sIVs contain more glycerophospholipids, further confirming that sIVs are intracellular components that mediate intracellular material transport and communication between organelles. However, sEVs contain more sphingomyelin. sEVs originate from invaginations of the cell membrane and are secreted to the extracellular space through the cell membrane, thus containing more components outside the cell membrane. This also indicates that sIVs lack an external membrane structure, and the differences in multiple lipids distinguish sIVs from sEVs. Previous studies have shown that the transport of proteins and lipids within cells is related to membrane curvature and lipid distribution effects. Glycerophospholipids can regulate membrane curvature and fluidity by adjusting chain length and cooperating with cholesterol, endowing sIVs with greater activity, thereby continuously participating in membrane fusion and fission events within the cell.

[0437] In this example, lipidomics data further verified that sIVs are different from sEVs, providing an important basis for in-depth exploration of the unique characteristics of sIVs in cell and tissue compatibility.

[0438] Example 8: Cells cultured in vitro and retinal tissue in vivo have higher absorption efficiency for sIVs

[0439] 1. Experimental instruments and materials

[0440] 1.1 Experimental Reagents

[0441] Table 9 Experimental reagents

[0442] 1.2 Experimental instruments

[0443] Table 10 Experimental instruments

[0444] 2. Experimental methods

[0445] 2.1 Cell culture

[0446] Human HRMECs were purchased from Angioproteomie, Inc., USA. Complete cell culture medium preparation: 5 ml of fetal bovine serum, 1 ml of growth factor, and 1 ml of penicillin-streptomycin were added to 93 ml of ECM basal medium to obtain a complete culture medium containing 5% serum. Complete RPE cell culture medium preparation: 5 ml of fetal bovine serum and 0.5 ml of penicillin-streptomycin were added to 44.5 ml of DMEM basal medium to obtain a complete culture medium containing 10% serum. Cell growth density was checked using a low-power microscope. High-power microscopy revealed round, oval, or polygonal flat cells with abundant cytoplasm and small intracellular vacuoles. Cells were seeded in culture flasks and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 3 days. When cell confluence reached 80%, cells were passaged at a 1:2 ratio, and experiments were performed using cells from P3 to P5.

[0447] 2.2 Experimental animals

[0448] Healthy male C57BL / 6 mice, 8 weeks old, weighing 21-22 g, SPF grade, were purchased from Beijing Weitonglihua Co., Ltd. (Animal Production License: SCXK (Beijing) 2016-0006). All experimental animals were fed a normal diet at room temperature and maintained under a 12-h light and dark cycle. The animal husbandry and experimental procedures complied with the Regulations on the Administration of Laboratory Animals of the State Science and Technology Commission of China and were approved by the Animal Ethics Committee of our institution (Ethics Number: TJYY2019091124). Mice were randomly assigned to groups for subconjunctival and intravitreal injections.

[0449] 2.3 DiD-labeled sIVs and sEVs co-cultured with cells

[0450] The vesicles were incubated with a lipid-soluble tracer solution of DiD at 37°C for 30 minutes. Ultra centrifugal filter to remove excess DiD. Cells were seeded into pre-placed circular pieces in 24-well culture plates and incubated for 24 hours. DiD-labeled vesicles were added to the cell culture medium and incubated at 37°C for different times: 3 hours, 12 hours, 24 hours or 48 hours. After removing the culture medium, the cells were washed with PBS at room temperature. The cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 for 5 minutes at room temperature to permeabilize the cells. Subsequently, the cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 at room temperature for 5 minutes to permeabilize the cells. The cells were then stained with a phalloidin antibody conjugated to Plus 488 for 30 minutes. DAPI was used to label the nuclei. Finally, cells were imaged using a confocal laser scanning microscope, and the amount of endocytosed vesicles was analyzed using Image J.

[0451] 2.4 Subconjunctival and intravitreal injection of DiD-labeled sIVs and sEVs

[0452] Vesicles were stained with DiD as described above. DiD-labeled vesicles were injected subconjunctivally into mice at a volume of 5 μL and a mass of 3 μg. Eyeballs were harvested for observation 24 and 48 hours after subconjunctival injection. DiD-labeled vesicles were injected into the vitreous cavity of mice at a volume of 1 μL and a mass of 1 μg. Eyeballs were harvested for evaluation 8 and 48 hours after intravitreal injection.

[0453] 2.5 Retinal Slices

[0454] Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. Retinal sections were fixed with PFA for 15 minutes at room temperature, then the slides were washed with PBS and the cell nuclei were stained with DAPI. Finally, the sections were mounted with anti-fluorescence attenuation mounting medium and observed using a confocal laser scanning microscope.

[0455] 2.6 Ultrasonic drug delivery

[0456] Rapa-sEVs and Rapa-sIVs were prepared by ultrasound. Rapa was mixed with sEVs or sIVs (prepared in Example 2) at a concentration ratio of 9:1. The mixture was incubated at room temperature for 10 minutes and then sonicated using an ultrasonic cytometer (25% power, 6 cycles, each cycle was a 30-second pulse and a 30-second pause). Subsequently, the mixture was incubated at 37°C for 1 hour to promote membrane recovery. After the ultrasound treatment process, free Rapa was removed by ultrafiltration centrifugation.

[0457] 2.7 Detection of Rapa in sIVs and sEVs by HPLC

[0458] High Performance Liquid Chromatography (HPLC) was used to assess the Rapa concentration in Rapa-sEVs or Rapa-sIVs. A standard curve was generated based on a range of Rapa concentrations (ranging from 0 to 50 μg / mL). 20 μL of standard and sample solutions were injected into the HPLC system for analysis. The standard curve was plotted using the average peak area as the abscissa and the Rapa concentration as the ordinate, and the regression equation was calculated. Acetonitrile was added to Rapa-sEVs or Rapa-sIVs to precipitate the proteins of sEVs or sIVs and extract Rapa. After centrifugation, Rapa was analyzed in the supernatant. The chromatographic column used was a C18 reverse-phase column. The mobile phase consisted of HPLC-grade acetonitrile and water (Vacetonitrile / Vwater = 65 / 35) with a flow rate of 1 ml / min. The detection wavelength was set to 278 nm and the column temperature was maintained at 62°C. The loading capacity (%) was calculated using the formula: (drug encapsulated volume) / (drug encapsulated volume + total mass of sEVs or sIVs) × 100%. The encapsulation efficiency (%) was calculated using the following formula: (drug encapsulated volume) / (total drug volume) × 100%.

[0459] 2.8 Detection of Rapa in the Retina by Liquid Chromatography-Mass Spectrometry

[0460] Precisely measure a 5mM Rapa standard and dissolve it in methanol to prepare a 50µM Rapa standard intermediate stock solution. Dissolve approximately 2mg of the internal standard carbamazepine standard in methanol to prepare a 2.00mg / ml carbamazepine standard stock solution. Precisely pipette an appropriate amount of the Rapa standard intermediate stock solution and serially dilute it with methanol to obtain Rapa working solutions with concentrations of 2000, 1000, 500, 200, 50.0, 20.0, 10.0, and 5.0nM, respectively. This will be used to prepare the standard curve samples for the dosing analysis batch. Transfer 50µl of methanol to a 1.5ml EP tube. Add 5µl of the standard curve working solution and 10µl of the internal standard working solution 2 to the tube. Vortex thoroughly to mix. Then, add 50µl of methanol and vortex to mix thoroughly before injecting the sample for analysis. The concentrations of Rapa standard curve in the sample analysis batch were: 200, 100, 50, 20, 5.0, 2.0, 1.0, and 0.5 nM. Frozen mouse retinal samples were removed, and 100 μL of chromatographic-grade methanol was added to each tube. Grinding beads were added and homogenized in a tissue grinder at 50 Hz for 2 minutes, with a 5-second pause every 30 seconds. 50 μL of the homogenate was transferred to a 1.5 ml EP tube. 5 μL of methanol solution and 10 μL of internal standard working solution 2 were added to the tube. After thorough vortexing, 50 μL of methanol was added to the extractant, vortexed for 1 minute, and centrifuged at 12,000 rpm for 10 minutes. 100 μL of the supernatant was sampled and analyzed. Mass spectrometry conditions: curtain gas: 25 psi, collision gas (CAD): Medium, ion source voltage (spray voltage): 5000 V, ion source temperature: 550° C., nebulizer gas (GS1): 60 psi, auxiliary heating gas (GS2): 55 psi.

[0461] Table 11 Liquid phase conditions

[0462] 3. Statistical processing

[0463] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0464] 4. Experimental results

[0465] 4.1 Cells have a better ability to internalize sIVs than sEVs

[0466] In order to evaluate the phagocytic ability of cells to two types of vesicles, RPE cells and HRMECs were selected to co-culture with MSC-sEVs (sEVs in this embodiment) and MSC-sIVs (sIVs in this embodiment). In short, an equal amount of DiD-labeled vesicles were incubated with cells, and at different time points, the distribution of DiD was observed using a confocal microscope to observe the cell uptake of vesicles. The results showed that after 3 hours of co-culture, cells began to internalize sEVs and sIVs, and over time, their uptake continued to increase (Figure 39A, Figure 39B, Figure 40A, Figure 40B). At 24 hours, the amount of sEVs and sIVs internalized by cells reached a peak, then gradually decreased within 24-48 hours (Figure 39C, Figure 40C). It is worth noting that in RPE cells and HRMECs, during 12 to 48 hours, the internalization rate of cells to sIVs always exceeded sEVs (Figure 39C, Figure 40C). This indicates that the cells cultured in vitro have high efficiency in absorbing sIVs.

[0467] 4.2 The retina has a better ability to internalize sIVs than sEVs

[0468] To evaluate the phagocytic ability of the retina for the two types of vesicles, equal amounts of DiD-labeled vesicles were administered to the eyes by two methods: subconjunctival injection and intravitreal injection. Eyeball samples were collected at different time points, and frozen sections were prepared and confocal microscopy was used to observe vesicle uptake in the retina. The results showed that 24 hours after subconjunctival injection, sIVs penetrated the sclera and entered the subretinal and retinal spaces, while sEVs accumulated between the conjunctiva and sclera, with fewer sEVs reaching the subretinal area (Figure 41A). After 48 hours, the retina showed significant uptake of sEVs and sIVs (Figure 41C). Specifically, sEVs were distributed in the RPE layer, while sIVs reached deeper locations and were widely distributed in the outer nuclear layer, inner nuclear layer and ganglion cell layer. After intravitreal injection of DiD-labeled vesicles, the vesicles spread from the vitreous cavity to the retina. The results showed that 8 hours after injection, sEVs were distributed in the vitreous cavity and had not yet reached the retina, while sIVs had reached the entire retinal layer and were internalized by cells in the ganglion cell layer and inner nuclear layer, with higher sIVs accumulation observed on the RPE layer (Figure 41B). 24 hours after intravitreal injection, the retina effectively absorbed sEVs and sIVs, and both types of vesicles were diffusely distributed throughout the retina (Figure 41B). Compared with sEVs, sIVs showed more significant uptake (Figure 41D) and were widely distributed in all retinal layers, including the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and RPE layer (Figure 41B).

[0469] 4.3 sIVs have better drug loading capacity than sEVs

[0470] To further investigate the retina's ability to absorb vesicle contents, we used ultrasound to encapsulate the lipophilic small molecule Rapa into vesicles. Transmission electron microscopy images showed that both sEVs and sIVs maintained their structural integrity after ultrasound treatment and drug loading (Figure 42A), without any significant change in vesicle size (Figure 42B). HPLC was used to assess the amount of Rapa carried by both types of vesicles after ultrasound treatment, with Figure 43A showing the characteristic peaks of Rapa. Calculations showed that sIVs exhibited significantly higher encapsulation and loading efficiencies compared to sEVs (Figures 43B and 43C).

[0471] 4.4 The retina has a stronger ability to absorb sIVs-loaded drugs

[0472] Based on the encapsulation and loading efficiency, the amount of Rapa carried by sEVs and sIVs was calculated. By supplementing with PBS, two vesicles carrying equal amounts of Rapa and the same amount of pure Rapa were obtained. The three solutions were then adjusted to equal volumes and injected into the subconjunctival space and vitreous cavity of mice, respectively. At different time points, retinal samples were collected to assess the Rapa content. The results showed that compared with direct injection of Rapa, the amount of Rapa absorbed by the retina from the vesicles was higher than that from pure Rapa 24 hours after subconjunctival injection of both drug-loaded vesicles. At 48 hours, the retina absorbed significantly more Rapa from sIVs than from sEVs, and this pattern continued until the fifth day (Figure 44A). At the same time, retinal samples collected 24 hours after intravitreal injection of both drug-loaded vesicles showed that sIVs delivered more Rapa to the retina than sEVs and pure Rapa (Figure 44B).

[0473] The above results show that sIVs have a larger drug-loading capacity, can carry more drugs, and the drugs carried by sIVs can be more absorbed and internalized by tissues. sIVs are good drug carriers.

[0474] 5. Summary

[0475] This example shows that cells cultured in vitro have better endocytosis ability for sIVs than sEVs. By performing subconjunctival injection and intravitreal injection on mice, it was observed that the retina has better endocytosis ability for sIVs than sEVs. This finding fully demonstrates that sIVs has good tissue compatibility. Subsequently, we used ultrasound drug loading technology to load the fat-soluble drug Rapa into sIVs and sEVs. The results showed that sIVs had a higher drug loading rate, and the vesicle-encapsulated Rapa increased the retina's absorption rate of Rapa. The retina has a higher absorption rate for Rapa carried by sIVs. This shows that sIVs are easily absorbed by tissues and have excellent ability to load fat-soluble small molecule drugs. The above results not only prove the feasibility of sIVs as a drug carrier, but also lay a solid foundation for its application in the treatment of posterior ocular diseases after loading drugs.

[0476] Example 9: Application of sIVs as a drug carrier for loading protein drugs

[0477] In this example, interleukin 10 (IL-10) was used as a drug example, and sIVs were further tested as a drug carrier.

[0478] 1. Experimental methods

[0479] 1.1 Ultrasound-mediated drug delivery of interleukin-10 (IL-10)

[0480] MSC-sIVs (prepared in Example 2, referred to as sIVs in this example) were added to the IL-10 solution in a gradient mass ratio (1:20, 1:10, 1:5, and 1:1). After mixing, the mixture was sonicated for 6 cycles of 30 seconds on / 30 seconds off. After sonication, the solution was passed through a 100 kDa ultrafiltration tube and centrifuged to remove free drug.

[0481] 1.2. Detection of IL-10-carrying efficiency of sIVs using enzyme-linked immunosorbent assay (ELISA)

[0482] IL-10-loaded sIVs were added to an ELISA kit plate, along with the corresponding standard control group. Incubate at room temperature for 1 hour. The appropriate volume of primary antibody was added, incubated at room temperature for 1 hour, and the plate was washed three times. The appropriate volume of secondary antibody was added, incubated at room temperature for 1 hour, and the plate was washed three times. The appropriate volume of substrate was added for color development. After incubation at room temperature for 20 minutes, the appropriate volume of stop solution was added to terminate color development. Absorbance was measured using a 562 microplate reader for excitation. Finally, the IL-10 expression level in each sample was calculated.

[0483] 1.3 T cell proliferation assay in vitro (CFSE method)

[0484] Pretreatment of 96-well plates (plating): Dilute CD3 antibody in PBS to a concentration of 10 μg / ml, add 100 μl to each well of the 96-well plate, and add 100 μl PBS to the outer wells of the 96-well plate to prevent excessive evaporation. Incubate at 4°C overnight or incubate in a cell incubator at 37°C for 2 hours. Aspirate the antibody, wash the wells twice with PBS and set aside.

[0485] Adjust Total CD4 + T cells were placed in a centrifuge tube to a volume of 5 ml, 1 μl of CFSE storage solution (5 mmol / L) was added, and the cells were quickly mixed and incubated at room temperature in the dark for 10 min. The same volume of RPMI1640 medium containing 10% FBS was added to terminate the staining. After centrifugation, the cells were resuspended in complete medium (configuration: 90% 1640 basal medium + 10% FBS + 2 μg / ml CD28 antibody + 100 U / ml double antibody + 50 μM β-mercaptoethanol) and the cells were plated at 3x10 5 The cells were plated in a pre-treated 96-well plate, and a blank control group and an IL-10-loaded sIVs-treated group were set up, with three replicate wells in each group. After 72 hours of culture, the cells were collected and stained with CD4-APC / CD40L-PerCp antibodies, and the expression of CFSE fluorescence and CD40L was detected by flow cytometry. + Changes in T cell proportions.

[0486] 2. Experimental results

[0487] 2.1 Drug loading efficiency of IL-10 loaded into sIVs

[0488] Pure IL-10 was loaded into sIVs by ultrasound. After ultrafiltration and washing, the IL-10 loaded sIVs were collected for ELISA testing. The results showed that the drug loading rate in three independent experiments was higher than 50% (Figure 45).

[0489] 2.3 IL-10-loaded sIVs have a significant inhibitory effect on T cell proliferation in vitro

[0490] IL-10-encapsulated sIVs were added to T cells cultured in vitro, and T cell proliferation was measured by flow cytometry. The results showed that IL-10-encapsulated sIVs significantly inhibited T cell proliferation in vitro compared to the control group (Figures 46 and 47).

[0491] 3. Summary

[0492] Combined with the experimental results of Example 7, it is shown that the sIVs prepared by the present invention has a good ability to load fat-soluble small molecule drugs and water-soluble proteins as a drug carrier, which can greatly improve the delivery efficiency.

[0493] Example 10: Protective effect of MSC-sIVs on blue light-induced retinal photodamage

[0494] 1. Experimental instruments and materials

[0495] 1.1 Experimental Reagents

[0496] Table 12 Experimental reagents

[0497] 1.2 Experimental Instruments

[0498] Table 13 Experimental instruments

[0499] 2. Experimental methods

[0500] 2.1 Experimental animals

[0501] BALB / c mice aged 6 weeks were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). The mice underwent a 16-hour dark adaptation period and were then exposed to 2000 Lux of blue light for 1 hour and then returned to darkness for another 16 hours. Before blue light irradiation, tropicamide (0.5%) was topically applied to the cornea to dilate the pupil. A box had a blue light on the top and mirrors on all four sides for light sensing, and each corner of the box could receive approximately 2000 Lux of illumination. Four hours before blue light irradiation, different concentrations of MSC-sEVs or MSC-sIVs (prepared in Example 2) (low concentration 2 μg and high concentration 4 μg) were injected intravitreally in a volume of 1 μL. The same amount of PBS was used as a negative control. Mice without blue light damage but injected with 1 μL of PBS were used as the normal group.

[0502] 2.2 Optical Coherence Tomography

[0503] Mice were anesthetized and their pupils dilated. Spectral domain OCT was performed using the Spectralis HRA+OCT device to examine retinal structural changes. Retinal thickness was measured using a circular scanning pattern centered on the optic nerve head (circle diameters 1, 3, and 6 ETDRS).

[0504] 2.3 Electroretinogram

[0505] BABL / c mice were dark-adapted for 12 hours 7 days after intravitreal injection and then ERG assessment was performed in the dark using a machine (Phoenix Research Labs, Pleasanton, CA). Mice were anesthetized using intraperitoneal injection. After anesthesia, metal electrodes were placed on the cornea, while reference electrodes were placed on the forehead and base of the tail. ERG measurements were obtained using a light stimulus consisting of a series of flash intensities [0.1, 1, and 2.2 Log (cd.s / m2)], each flash lasting 1ms. The flashes were used to induce photoreceptor cell (a wave) and bipolar cell responses (b wave). Recordings were made after each flash; the three responses were averaged with an interstimulus interval of 1s (for 0.1Log (cd.s / m2)), 10s (for 1Log (cd.s / m2)), or 30s (for 2.2Log (cd.s / m2)), and the amplitude (μV) was recorded.

[0506] 2.4 Histological evaluation of mouse retina

[0507] BABL / c mice were sacrificed by cervical dislocation 7 days after intravitreal injection and their eyeballs were collected. The collected eyeballs were fixed in 4% paraformaldehyde and embedded in paraffin. Subsequently, 4 μm horizontal sections of the retina were prepared from the paraffin-embedded samples and stained with hematoxylin and eosin. Images of each section of a single eye were captured using a microscope (Zeiss, Germany).

[0508] 2.5 Immunofluorescence staining

[0509] BABL / c mice were killed by cervical dislocation 7 days after intravitreal injection and the eyeballs were collected. Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. PFA was used to fix the slides at room temperature for 15 minutes, and the slides were then washed with PBS. The slides were incubated in blocking buffer (10% normal goat serum) at room temperature for 1 hour. The primary antibody was applied to the slides and incubated at 4°C overnight. The slides were then washed with PBS and incubated with a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 488 (dilution 1:5000) at room temperature for 2 hours. After incubation, the slides were rinsed with PBS and the cell nuclei were stained with DAPI. Finally, the slides were sealed with anti-fluorescence attenuation mounting medium and observed using a confocal microscope.

[0510] 2.6 Quantification of retinal ganglion cells and microglia

[0511] BABL / c mice were killed by cervical dislocation 7 days after intravitreal injection and the eyeballs were collected. The eyeballs were removed and fixed in 4% paraformaldehyde for 30 minutes. The retina was carefully dissected, rinsed with PBS, and then permeabilized with PBS containing 1% Triton X-100 for 30 minutes. Then, the retina was blocked with a PBS solution containing 2% BSA and 0.3% Triton X-100 at 4 ° C for 2 hours. The flat-mounted retinas were then stained with Rbpms and Iba1 antibodies and incubated overnight in the dark at 4 ° C to observe retinal ganglion cells and microglia. The retina was observed using a confocal fluorescence microscope. As described in the literature (Gharagozloo M., Smith MD, Jin J., et al. Complement component 3 from astrocytes mediates retinal ganglion cell loss during neuroinflammation [J]. Acta Neuropathol, 2021, 142 (5): 899-915), the retina was divided into regions. ImageJ software was used to quantify the number of retinal ganglion cells and microglia.

[0512] 3. Statistical processing

[0513] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0514] 4. Experimental results

[0515] MSC-sIVs protect retinal structure from blue light damage

[0516] Blue light exposure can cause structural damage to the retina, mainly manifested as retinal thinning. MSC-sEVs and MSC-sIVs were used to treat blue light-induced retinal damage, and changes in retinal structure and function were evaluated using OCT and ERG. The results showed that retinal thickness was significantly reduced after blue light exposure (Figure 48). Low doses of MSC-sEVs and MSC-sIVs did not show significant therapeutic effects on retinal thinning; however, high doses of MSC-sEVs and MSC-sIVs significantly improved retinal thinning (Figure 48C).

[0517] Eyeballs of mice with retinal damage induced by blue light irradiation (abbreviated as BL in this example) were collected 7 days later. H&E staining showed that the outer nuclear layer (ONL) of BL mice treated with PBS was significantly thinner and a large number of cells were lost (Figure 49A), and the inner and outer fragments of photoreceptor cells and most of the cell nuclei in the ONL layer were lost (Figure 49A). This degeneration was greatly reduced in BL mice treated with MSC-sIVs (Figure 49A). Quantification of ONL cell nucleus counts in retinal sections confirmed the extensive loss of photoreceptor cells due to degeneration in PBS-treated mice, and confirmed that retinal damage was alleviated in BL mice treated with high-dose sEVs and low-dose and high-dose sIVs (Figure 49B).

[0518] MSC-sIVs protect retinal electrophysiological function from blue light damage

[0519] To further investigate the role of high-dose MSC-sEVs and MSC-sIVs in improving retinal electrophysiological function, we used ERG to detect retinal function in mice. The average amplitudes of ERG a and b waves in BL mice treated with PBS were significantly reduced after seven days of blue light exposure. Figure 50A shows a representative ERG waveform of one retina in each treatment group. Under high-intensity light stimulation, BL mice treated with PBS essentially lost their photoreceptor response, and the waveform was extinguished. In the MSC-sEVs-treated group, the retina partially recovered its response to light, while the MSC-sIVs-treated group showed significantly improved retinal light response (Figure 50B).

[0520] MSC-sIVs reduce blue light-induced retinal apoptosis and photoreceptor cell damage

[0521] Retinal sections from mice sacrificed after seven days of blue light induction were collected and stained with TUNEL and DAPI to mark nuclei with double-stranded DNA breaks (markers of programmed cell death or other forms of cell death). Representative images showed that apoptosis signals were mainly concentrated in the outer nuclear layer of the retina (Figure 51A). The retinas of BL mice treated with PBS showed high levels of apoptosis signals, while the BL mice treated with MSC-sEVs and MSC-sIVs showed reduced apoptosis signals. The high-dose MSC-sIVs group showed the most significant reduction in apoptosis (Figure 51B).

[0522] To further explore the distribution of photoreceptor cells and synaptic connections in the retina, retinal sections were stained with Rhodopsin, a specific marker for photoreceptor cells. As shown in representative images (Figure 52A), rhodopsin protein is mainly present in the rod photoreceptor cells of the retina, especially in the outer layer of these cells, and plays a vital role in phototransduction, that is, converting light into electrical signals. In PBS-treated BL mice, the distribution of rhodopsin protein was limited to the outer layer of a few photoreceptor cells in the retina, while BL mice treated with MSC-sEVs and MSC-sIVs showed larger rhodopsin protein-positive areas (Figure 52B). Among them, high-dose MSC-sIVs treatment showed the highest degree of prevention of rhodopsin loss compared with the same dose of MSC-sEVs (Figure 52B). Western blot analysis of retinal total protein extracts further supported the above fluorescence quantification results (Figure 53). These findings indicate that MSC-sIVs treatment can more effectively reduce photoreceptor cell apoptosis and improve synaptic connections than MSC-sEVs treatment.

[0523] MSC-sIVs reduce blue light-induced retinal inflammation activation

[0524] Exposure to intense blue light causes oxidative stress and inflammation, leading to retinal cell death and subsequent visual impairment. GFAP is a marker of activated Müller cells, indicating the presence of retinal inflammation. Immunofluorescence staining for GFAP was performed on ocular sections of mice euthanized seven days after blue light exposure. In ocular sections of normal mice, GFAP staining was primarily confined to the vicinity of the internal limiting membrane, with less staining in the outer plexiform layer (Figure 54A). In the eyes of mice exposed to blue light and treated with PBS, significant GFAP staining across the inner plexiform layer was observed (Figure 54A). This additional signal was attenuated in mice exposed to blue light but treated with MSC-sEVs and MSC-sIVs (Figure 54A). Quantification of GFAP signal confirmed a statistically significant increase in GFAP expression in the retina of BL mice, but treatment with both low and high doses of MSC-sEVs and MSC-sIVs prevented this increase, with treatment with high doses of MSC-sIVs producing the most significant effect (Figures 54A and 54B). Western blot analysis of total retinal protein extracts further supported the above fluorescence quantification results (Figure 53).

[0525] To further evaluate the effect of MSC-sIVs treatment on retinal inflammation in BL mice, Iba1 was used to label microglia in mouse retinal flat mounts, and cell counts were performed in the inner and outer layers of the retina. In mice not exposed to blue light, neatly arranged and regularly shaped Iba1-positive cells were detected in the inner layer of the retina, while a small number of Iba1-positive cells were found in the outer layer of the retina (Figure 55A). However, a large number of Iba1-positive cells were found in the retinas of mice irradiated with blue light, arranged in an irregular pattern. This increase was inhibited to varying degrees in the MSC-sEVs and MSC-sIVs treatment groups. Cell counting showed that the same dose of sIVs had a more significant inhibitory effect than sEVs (Figures 55B, 55C, and 55D). These data indicate that blue light exposure leads to the appearance of microglia-positive cells in the outer layer of the retina, and MSC-sIVs largely inhibits the increase in microglia.

[0526] 5. Summary

[0527] In this example, we used MSC-sEVs and MSC-sIVs to simultaneously treat a mouse model of blue-light-induced retinopathy. The results showed that MSC-sIVs could slow retinal thinning, with greater efficacy at higher doses. MSC-sIVs were superior to MSC-sEVs in slowing nuclear loss and improving electrophysiological function. Furthermore, high-dose MSC-sIVs significantly reduced retinal inflammation and apoptosis. Our study demonstrates the protective effect of MSC-sIVs in a mouse model of retinal light damage.

[0528] Example 11: Protective effect of MSC-sIVs on retinal degeneration in rd10 mice

[0529] 1. Experimental instruments and materials

[0530] 1.1 Experimental Reagents

[0531] Table 14 Experimental reagents

[0532] 1.2 Experimental instruments

[0533] Table 15 Experimental instruments

[0534] 2. Experimental methods

[0535] 2.1 Experimental animals

[0536] rd10 mice were provided by Professor Xu Haiwei of Southwest Medical University, and C57BL / 6J (wild type, WT) control mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). 14-day-old rd10 mice were treated with MSC-sEVs, MSC-sIVs, or PBS (control group). The animals were housed under standard, specific pathogen-free (SPF) conditions in the laboratory animal department with a 12-h light / dark cycle, with 4-5 mice per cage. The mice in this study were maintained on a standard normal diet. All procedures involving animals were performed in accordance with the ARVO instructions for the use of animals in ophthalmic and visual research and the Guidelines for Animal Research of the Institute of Ophthalmology, Tianjin Medical University.

[0537] 2.2 Intravitreal injection in mice

[0538] Intravitreal injection was performed in rd10 mice at p12. Tropicamide compound was used to dilate the pupil, lidocaine hydrochloride was used to anesthetize the ocular surface, and a 33G microsyringe was used for intravitreal injection. MSC-sEVs and MSC-sIVs (prepared in Example 2) were injected into the vitreous cavity in a volume of 1 μL and a mass of 2 μg. The same volume of PBS was used as a control.

[0539] 2.3 Electroretinogram

[0540] Same as 2.3 of Example 10.

[0541] 2.4 Histological evaluation of mouse retina

[0542] Same as 2.4 of Example 10.

[0543] 2.5 Immunofluorescence staining

[0544] Same as 2.5 of Example 10.

[0545] 2.6 Quantification of retinal microglia

[0546] Same as 2.6 of Example 10.

[0547] 3. Statistical processing

[0548] Same as 3 of Example 10.

[0549] 4. Experimental results

[0550] 4.1 Protective effect of MSC-sIVs on retinal outer nuclear layer cells in rd10 mice

[0551] To evaluate the protective effects of MSC-sIVs, we initially used H&E staining to visualize the structure and cell number of the ONL in the retina at p28. At p28, ONL cells in rd10 mice were almost completely lost (Figure 56A). However, both MSC-sEVs and MSC-sIVs treatment were able to reverse these changes, with MSC-sIVs demonstrating a significantly more pronounced therapeutic effect compared to MSC-sEVs (Figure 56B).

[0552] 4.2 Protective effect of MSC-sIVs on retinal function in rd10 mice

[0553] We used ERG to assess retinal electrophysiological function in rd10 mice at p28. At p28, the ERG waveforms of untreated rd10 mice exhibited a quenched state, representing a complete collapse of photoreceptor cell function (Figure 57A). Light responses in rd10 mice were significantly reduced at all tested flash intensities (Figure 57B), and treatment with MSC-sEVs and MSC-sIVs significantly rescued the light responses in rd10 retinas (Figure 57B). Statistically, MSC-sEVs significantly increased a- and b-wave amplitudes at moderate to high stimuli (>1.0 cd·s / m²), while MSC-sIVs significantly enhanced a- or b-wave amplitudes at low, moderate, and high stimuli (>0.1 cd·s / m²). Furthermore, MSC-sIVs significantly improved a-wave amplitude at the highest stimulus and b-wave amplitude at low, moderate, and high stimuli (>0.1 cd·s / m²) compared to MSC-sEVs. In conclusion, our results indicate that MSC-sIVs enhance visual function in rd10 mice.

[0554] 4.3 Inhibitory effect of MSC-sIVs on retinal apoptosis in rd10 mice

[0555] At p28, retinal sections of rd10 mice were collected and stained with TUNEL and DAPI to mark cell nuclei with double-stranded DNA breaks. Representative images showed that apoptotic signals were mainly concentrated in the outer nuclear layer of the retina (Figure 58A). In contrast, rd10 mice treated with MSC-sEVs and MSC-sIVs showed reduced apoptotic signals, with the most significant reduction in apoptosis observed in the MSC-sIVs treatment group compared to the MSC-sEVs treatment group (Figure 58B).

[0556] 4.4 Protective effect of MSC-sIVs on retinal photoreceptor cells in rd10 mice

[0557] Rhodopsin staining was used to further examine the photoreceptor cells of the retina. In rd10 mice, the loss of photoreceptor cells in the retina resulted in reduced rhodopsin protein expression (Figure 59). However, compared with MSC-sEVs, MSC-sIVs showed a more significant therapeutic effect (Figure 59).

[0558] At the same time, to further investigate the effects of MSC-sIVs on synaptic connections in the retina of rd10 mice, retinal sections were stained with PSD95. PSD95 is a postsynaptic protein that is located in the presynaptic terminals of photoreceptor neurons in the retina. In the rd10 retina, significantly weaker staining and loss of the band structure of PSD95 expression were observed (Figure 60A). However, both MSC-sEVs and MSC-sIVs treatment were able to rescue the loss of PSD95 expression, and the recovery effect of MSC-sIVs was significantly better than that of MSC-sEVs (Figure 60B).

[0559] 4.5 Inhibitory effect of MSC-sIVs on inflammation in rd10 mice

[0560] We used immunostaining to evaluate the expression of GFAP in the retinas of WT and rd10 mice. As previously mentioned, GFAP is mainly expressed in astrocytes located in the ganglion cell layer of the normal retina. In contrast, in the rd10 group, GFAP staining extended along the Müller glia throughout the retina (Figure 61A). MSC-sEVs and MSC-sIVs treatment reduced the expression of GFAP in Müller cells. Quantification of GFAP fluorescence intensity showed that the MSC-sIVs treatment group exhibited a significantly greater therapeutic effect compared to the MSC-sEVs treatment group (Figure 61B). Western Blot was used to further confirm the above fluorescence quantitative analysis results (Figure 64).

[0561] The activity state of microglia was detected by Iba1 staining. In rd10 mice, Iba1-positive cells in the retina were found to be disordered, and the number of inner and outer retinal layers increased (Figure 62A). Iba1-positive cells were observed in all layers of the rd10 retina, especially in the ONL and outer segments, where microglia exhibited a reactive morphology (characterized by enlarged cell bodies and retracted protrusions) (Figure 62A, Figure 63). Treatment with MSC-sEVs and MSC-sIVs significantly reduced the number of Iba1-positive cells in the inner and outer retinal layers. Specifically, MSC-sIVs were superior to sEVs in reducing the number of Iba1-positive cells in the outer retina and the entire retina (Figure 62B).

[0562] 5. Summary

[0563] This example used both MSC-sEVs and MSC-sIVs to treat rd10 mice. Compared to MSC-sEVs, MSC-sIVs significantly slowed retinal photoreceptor cell loss caused by retinal degeneration. MSC-sIVs also reduced retinal photoreceptor apoptosis and microglial activation.

[0564] Example 12: MSC-sIVs improve retinal damage by inhibiting endoplasmic reticulum stress

[0565] 1. Experimental instruments and materials

[0566] 1.1 Experimental Reagents

[0567] Table 16 Experimental Reagents

[0568] 1.2 Experimental instruments

[0569] Table 17 Experimental instruments

[0570] 2. Experimental methods

[0571] 2.1 Retinal protein extraction

[0572] For Babl / c mice, 7 days after blue light exposure, mice were killed by cervical dislocation, and the eyeballs and retinas were removed and placed in EP tubes, snap-frozen in liquid nitrogen, and stored at -80°C. For rd10 mice, mice were killed by cervical dislocation on p21 and p28, and the eyeballs and retinas were removed and placed in EP tubes, snap-frozen in liquid nitrogen, and stored at -80°C.

[0573] Add 150 μL of Ripa protein lysis buffer containing protease inhibitors to each retina. Use a pipette tip to perforate the retina and sonicate. Sonicate on ice at 35% power, cycle on for 2 seconds, then off for 2 seconds for a total of 1 minute. Centrifuge at 12,000 g for 15 minutes at 4°C. Collect the supernatant as the retinal protein.

[0574] 2.2 Retinal mRNA-seq

[0575] 2.2.1 RNA extraction and library construction

[0576] RNA integrity and total quantity were assessed using an Agilent 2100 bioanalyzer to ensure high sample quality. Libraries were constructed using total RNA. Oligo(dT) magnetic beads were used to enrich mRNA with poly(A) tails, which were then randomly fragmented using divalent cations in a specific fragmentation buffer. First-strand cDNA was synthesized using these fragmented mRNAs as templates and random oligonucleotides as primers using an M-MuLV reverse transcriptase system. RNase H was then used to degrade the RNA strands, and second-strand cDNA was synthesized using dNTPs in a DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and ligated with sequencing adapters. cDNA fragments of approximately 370 to 420 bp were selected using AMPure XP beads, amplified by PCR, and purified again using AMPure XP beads to generate the desired library.

[0577] After library construction, initial quantification was performed using a Qubit 2.0 Fluorometer, diluting the library to 1.5 ng / μl. The insert size of the library was then measured using an Agilent 2100 Bioanalyzer to ensure it met expectations. Finally, qRT-PCR was used to accurately determine the effective concentration of the library (ensuring it was above 2 nM) to ensure high library quality.

[0578] 2.2.2 Sequencing

[0579] Sequencing was performed using the Illumina NovaSeq 6000, generating 150bp paired-end reads. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. Each time the sequencing cluster extends the complementary strand and incorporates a fluorescently labeled dNTP, a corresponding fluorescent emission is released. The sequencer captures these fluorescent signals and uses computer software to convert the optical signals into sequencing peaks, thereby obtaining sequence information for the fragment being tested.

[0580] 2.2.3 Data processing and analysis

[0581] The image data generated by the high-throughput sequencer is converted into sequence data (reads) after base recognition processing by CASAVA. The raw data may contain some reads with sequencing adapters or low quality, which need to be filtered to ensure the accuracy and reliability of the data analysis. The filtering steps mainly include: removing reads with adapters, eliminating reads with uncertain base information (N), and removing low-quality reads (i.e., reads with bases with Qphred values ​​​​less than or equal to 20 accounting for more than 50% of the total read length). At the same time, the Q20, Q30 values ​​and GC content of the filtered data (clean data) are calculated. All subsequent analyses are based on this cleaned data. The reference genome and gene model annotation files are obtained directly from authoritative genome websites. HISAT2 (v2.0.5) is used to construct an index of the reference genome, and the cleaned paired-end reads are aligned with the reference genome. HISAT2 was chosen as the alignment tool because it can generate a spliced ​​database based on gene model annotation files, thus providing more accurate alignment results than other non-spliced ​​alignment tools.

[0582] 2.2.4 Quantitative analysis of gene expression

[0583] FeatureCounts (1.5.0-p3) was used to count the number of reads mapped to each gene. Subsequently, the FPKM value (the expected number of fragments per kilobase per million base pairs of transcript sequence fragments) was calculated for each gene based on its length. This method, which accounts for the effects of both sequencing depth and gene length on read counts, is currently one of the most commonly used methods for assessing gene expression levels.

[0584] 2.2.5 Differential expression analysis

[0585] Differential expression analysis was performed using DESeq2 software (1.20.0). DESeq2 determines differential expression in digital gene expression data based on a negative binomial distribution model and provides corresponding statistical procedures. P values ​​(padj) were adjusted by the method of Benjamini and Hochberg to control the false discovery rate. The threshold for significant differential expression was set at padj < = 0.05 and |log2(foldchange)| > = 1.

[0586] 2.2.6 Enrichment analysis of differentially expressed genes

[0587] ClusterProfiler (3.8.1) software was used to perform GO enrichment analysis on differentially expressed genes, correcting for gene length bias. GO terms with corrected P values ​​less than 0.05 were considered significantly enriched for significantly differentially expressed genes. ClusterProfiler was also used to analyze the statistical enrichment of differentially expressed genes in KEGG pathways, Reactome pathways, DO pathways, and DisGeNET pathways. These database resources facilitate molecular understanding of the high-level functions and utilities of biological systems, such as cells, organisms, and ecosystems. The significant enrichment threshold for KEGG, Reactome, DO, and DisGeNET was set at a corrected P value less than 0.05.

[0588] 2.3 Western Blot

[0589] Same as 2.4 of Example 4.

[0590] 3. Statistical processing

[0591] Same as 3 of Example 10.

[0592] 4. Experimental results

[0593] 4.1 RNA-seq results show the therapeutic effect of MSC-sIVs on light-damaged retina

[0594] To explore the therapeutic effect of MSC-sIVs on the retinas of light-damaged mice, we collected the retinas of light-damaged mice 7 days after MSC-sIVs treatment (from Example 10) and performed RNA sequencing on the retinas of untreated (PBS-injected) light-damaged mice. The results showed significant differences in the following pathways: folate-mediated one-carbon unit metabolic pool, calcium signaling pathway, vascular smooth muscle contraction, cGMP-PKG signaling pathway, thiamine metabolism, cofactor biosynthesis, myocardial contraction and oxytocin signaling pathway, etc. (Figure 65).

[0595] RNA-seq results show the therapeutic effect of MSC-sIVs on retinal degeneration in rd10 mice

[0596] To explore the therapeutic effect of MSC-sIVs on retinal damage in rd10 mice, we collected the retinas of rd10 mice (from Example 11) 7 days after MSC-sIVs treatment and performed RNA-seq on the retinas of untreated (PBS-injected) rd10 mice. The results showed significant differences in the following pathways: L1-mediated vesicle transport recycling pathway, L1CAM interaction, IRE1alpha activation of chaperone proteins, XBP1(S) activation of chaperone protein genes, unfolded protein response (UPR), activation of mRNA after cap binding complex and eIFs binding, cell-extracellular matrix interaction, transport to the Golgi apparatus and its subsequent modification, COPI-mediated forward transport, forward transport from the endoplasmic reticulum to the Golgi apparatus, and axon guidance, etc. (Figure 66).

[0597] 4.3 Proteomic analysis showed that MSC-sIVs could negatively regulate ER stress

[0598] This study confirmed that MSC-sIVs have a better therapeutic effect than MSC-sEVs at the same dose in blue light-damaged retina and rd10 mouse retina (see Examples 10 and 11). To further explore the role of biomacromolecules contained in the two vesicles in regulating cellular metabolism, we performed Gene Set Variation Analysis (GSVA) ​​pathway enrichment analysis on the total proteins contained in the two. The results showed that the following pathways were significantly enriched in MSC-sIVs: negative regulation of endoplasmic reticulum stress response, endoplasmic reticulum component secretory granules, clathrin-coated vesicles, transport vesicles between the endoplasmic reticulum and the Golgi apparatus, transport from the Golgi apparatus to the plasma membrane, CopI vesicles and CopII vesicles. Cell-to-cell adhesion, cell-to-matrix adhesion and substrate adhesion were significantly enriched in MSC-sEVs (Figure 67).

[0599] 4.4 miRNA-seq analysis showed that MSC-sIVs can regulate endoplasmic reticulum homeostasis

[0600] To further analyze the role of MSC-sIVs in improving retinal damage compared to MSC-sEVs, we extracted the top 50 miRNAs expressed in sEVs and sIVs of MSCs cells (Figure 68), performed differential analysis, and selected 24 differential miRNAs for target gene enrichment analysis. The results are shown in Figure 69. These 24 miRNAs can regulate the following pathways: endoplasmic reticulum calcium homeostasis, COPI vesicle envelope, protein tyrosine kinase activity, protein kinase B signaling, collagen catabolism, positive regulation of STAT protein tyrosine phosphorylation, negative regulation of gene expression through CpG island methylation, negative regulation of endopeptidase activity, positive regulation of mitotic spindle microtubules and centromere side connection, DNA methyltransferase activity, apical cortical central nervous system morphogenesis, etc.

[0601] Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in light-damaged mice

[0602] In Examples 4.3 and 4.4 of this study, we revealed that MSC-sIVs can act on the calcium ion signaling channels of the retina of light-damaged mice, and also on the endoplasmic reticulum stress signaling pathways (UPR, XBP1, and IRE1α) of the retina of rd10 mice. On the other hand, in an in-depth analysis of the proteins and miRNAs of MSC-sIVs and MSC-sEVs, it was found that MSC-sIVs had a negative regulatory effect on endoplasmic reticulum stress and could maintain endoplasmic reticulum homeostasis. Therefore, we used Western blot to further detect the expression of endoplasmic reticulum stress-related proteins in the retina under light damage conditions. Glucose-regulated protein 78 (GRP78) binds to three endoplasmic reticulum stress sensors, and upregulated GRP78 expression is one of the most commonly used markers of endoplasmic reticulum stress. Therefore, we examined GRP78 by Western blot analysis in mouse retinas before, and 3, 5, and 7 days after blue light irradiation. The results showed that GRP78 expression levels increased from 3 to 7 days after irradiation compared to unirradiated mice, with a more pronounced increase on day 5 (Figure 70). These results suggest that excessive blue light exposure causes ER stress in the mouse retina, and ER stress may be a therapeutic target for protecting the retina from light damage.

[0603] After confirming the presence of ER stress in the retina of blue light-damaged mice, we used Western Blot to detect related target proteins and explored the effects of different doses of MSC-sEVs and MSC-sIVs on the ER stress pathway. The results showed that compared with the NoBL group, the expression of retinal ER stress-related proteins in the light-damaged group (BL-PBS) treated with PBS was increased, as shown by increased levels of GRP78, p-PERK / PERK, IRE1a, ATF6, p-eIF2a and CHOP, and the differences were statistically significant (p<0.05; Figure 71); compared with the BL-PBS group, there was no significant difference in the expression levels of ER stress-related proteins in the low-dose and high-dose MSC-sEVs treatment groups; however, compared with the BL-PBS group The low-dose MSC-sIVs treatment group showed a decrease in the expression levels of some ER stress-related proteins, as evidenced by a decrease in the expression levels of p-PERK / PERK and IRE1a, with statistically significant differences (p<0.05). The high-dose MSC-sIVs treatment group also showed a decrease in the expression levels of ER stress-related proteins, as evidenced by a decrease in the expression levels of GRP78, p-PERK, ATF6, IRE1a, p-eIF2a / eIF2a, and CHOP, with statistically significant differences (p<0.05, Figure 71). At the same time, the same dose of sIVs significantly inhibited the expression levels of GRP78 and IRE1a compared to sEVs (p<0.05, Figure 71). These results suggest that excessive blue light exposure causes ER stress in the retina, and MSC-sIVs protect the retina from light damage by inhibiting ER stress.

[0604] ER stress ultimately leads to cell apoptosis. Next, we examined the expression levels of apoptosis-related proteins in the retina. The results showed that compared with the NoBL group, BL-PBS cells showed an increase in apoptosis-related proteins, as evidenced by increased expression of Cleaved-Caspase3 and Bax, and decreased expression of the anti-apoptotic protein Bcl-2 (p < 0.05; Figure 72). There was no significant difference in the expression of apoptosis-related proteins between the low- and high-dose MSC-sEVs-treated groups compared with the BL-PBS group. However, compared with the BL-PBS group, the high-dose MSC-sIVs-treated group showed decreased expression of apoptosis-related proteins, as evidenced by decreased expression of Cleaved-Caspase3, decreased expression of the pro-apoptotic protein Bax, and increased expression of the anti-apoptotic protein Bcl-2 (p < 0.05, Figure 72). The same dose of MSC-sIVs significantly increased the expression of the anti-apoptotic protein Bcl-2 compared with MSC-sEVs. These results suggest that excessive blue light exposure causes retinal apoptosis and MSC-sIVs can protect the retina from light damage.

[0605] 4.6 Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in rd10 mice (p21)

[0606] We further examined the expression of ER stress-related proteins in the retina of rd10 mice at different developmental stages using Western blot. We examined the expression of GRP78 in the retinas of rd10 mice at 14, 21, and 28 days of age without intervention. The results showed that GRP78 expression levels began to increase in the retina at P14 compared to WT mice of the corresponding ages, with more significant increases at P21 and P28 (Figure 73). These results suggest that ER stress occurs in the retina of rd10 mice and may represent a potential therapeutic target for protecting the retina of rd10 mice.

[0607] After confirming the presence of ER stress in rd10 mice, we investigated the effects of different doses of MSC-sEVs and MSC-sIVs on the ER stress pathway by Western blotting to detect relevant target proteins. The results showed that compared with the WT group, the expression of ER stress-related proteins in the retina of p21 rd10 mice was elevated, with statistically significant increases in GRP78, IRE1a, ATF6, and CHOP (p < 0.05; Figure 74). Compared with the rd10-PBS group, only ATF6 expression was decreased in the retinal ER stress-related proteins treated with MSC-sEVs; the expression levels of the other markers remained unchanged. However, the expression levels of ER stress-related proteins were decreased in the MSC-sIVs group, with statistically significant decreases in GRP78, IRE1a, ATF6, and CHOP (p < 0.05; Figure 74). These results suggest that MSC-sIVs reduce retinal damage in rd10 mice by inhibiting ER stress.

[0608] We examined the expression levels of apoptosis-related proteins in the retina of rd10 mice. The results showed that compared with the WT group, the expression of apoptosis-related proteins in the retina of rd10 mice at p21 was increased, as evidenced by increased expression of Cleaved-Caspase3 and Bax, and decreased expression of the anti-apoptotic protein Bcl-2. The differences were statistically significant (p < 0.05; Figure 75). Compared with the rd10-PBS group, the expression level of Cleaved-Caspase3 was decreased in the MSC-sEVs-treated group, but the expression level of the pro-apoptotic protein Bax remained high, while the expression level of the anti-apoptotic protein Bcl-2 did not increase significantly. On the other hand, the expression levels of apoptosis-related proteins in the retina of MSC-sIVs-treated group were decreased, as evidenced by decreased expression of Cleaved-Caspase3 and the pro-apoptotic protein Bax, and increased expression of the anti-apoptotic protein Bcl-2. The differences were statistically significant (p < 0.05, Figure 75). Compared to MSC-sEVs, the same dose of MSC-sIVs significantly reduced Caspase-3 activation and the expression of the pro-apoptotic protein Bax, while increasing the expression of the anti-apoptotic protein Bcl-2. These results suggest that retinal apoptosis in rd10 mice at p21 is significantly better than that of MSC-sEVs in reducing retinal apoptosis.

[0609] 4.7 Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in rd10 mice (p28)

[0610] Figure 73 confirmed that rd10 mice had ER stress at the p28 time point. We harvested mouse retinas at p28 and continued to detect ER stress-related target proteins by Western Blot to explore the effects of MSC-sEVs and MSC-sIVs on the ER stress pathway. The results showed that compared with the WT group, the expression of ER stress-related proteins in rd10 mice at p28 was generally increased, as shown by increased expression levels of GRP78, p-PERK / PERK, IRE1a, ATF6, p-eIF2a / eIF2a, and CHOP, with statistically significant differences (p<0.05; Figure 76). There was no significant difference in the expression levels of ER stress-related proteins in the retina of the MSC-sEVs-treated group compared with the rd10-PBS group. However, the expression levels of ER stress-related proteins in the MSC-sIVs treatment group were decreased, as shown by decreased expression levels of GRP78, p-PERK / PERK, IRE1a, ATF6, p-eIF2a / eIF2a, and CHOP, with statistically significant differences (p<0.05; Figure 76). These results indicate that MSC-sIVs can reduce retinal damage in rd10 mice by inhibiting ER stress, with a significantly better effect than MSC-sEVs.

[0611] We examined the expression levels of apoptosis-related proteins in the retina of rd10 mice. The results showed that compared with the WT group, rd10 mice at p28 had increased expression of apoptosis-related proteins in the retina, as evidenced by increased expression of cleaved-Caspase3 and Bax, and decreased expression of the anti-apoptotic protein Bcl-2. The differences were statistically significant (p < 0.05; Figure 77). MSC-sEVs treatment had no significant therapeutic effect on apoptosis-related proteins compared with the rd10-PBS group. The expression levels of apoptosis-related proteins in the retina of MSC-sIVs treatment were decreased, as evidenced by decreased expression of cleaved-Caspase3, decreased expression of the pro-apoptotic protein Bax, and increased expression of the anti-apoptotic protein Bcl-2. The differences were statistically significant (p < 0.05, Figure 77). Compared with MSC-sEVs, the same dose of MSC-sIVs significantly reduced Caspase3 activation and the expression of the pro-apoptotic protein Bax, while increasing expression of the anti-apoptotic protein Bcl-2. The above results indicate that the retina of rd10 mice undergoes apoptosis at p28, and MSC-sIVs can inhibit the apoptotic signal of the retina of rd10 mice and promote the expression of anti-apoptotic proteins, and its effect is significantly better than MSC-sEVs.

[0612] 5. Summary

[0613] In this example, we further explored the molecular mechanisms of MSC-sIVs in treating retinal injury. Through RNA-seq analysis of the retina, we found enrichment of the calcium signaling pathway in the retinas of light-damaged mice, while significant enrichment of the endoplasmic reticulum stress signaling pathway was observed in the retinas of rd10 mice. To further reveal the protein expression profile of MSC-sIVs, we employed GSVA, and the results showed that MSC-sIVs negatively regulate endoplasmic reticulum stress. By enrichment analysis of highly abundant miRNAs in MSC-sIVs, we found that these miRNAs play a key role in regulating endoplasmic reticulum homeostasis. In subsequent experiments, we observed significantly elevated expression of endoplasmic reticulum stress signaling in the retinas of light-damaged mice and rd10 mice. To validate the therapeutic efficacy of MSC-sIVs, we used Western blotting to examine the expression levels of endoplasmic reticulum stress signaling in the retinas after treatment. The results demonstrated that MSC-sIVs significantly inhibited endoplasmic reticulum stress signaling and effectively reduced retinal apoptosis.

[0614] When the ER faces stress, such as disruptions in protein synthesis, folding, or transport, a series of signaling pathways are activated to restore cellular homeostasis or induce apoptosis. Under stressful conditions, calcium ions are released from the ER into the cytoplasm, triggering calcium-dependent signaling. Simultaneously, the IRE1α protein on the ER membrane is activated, cleaving XBP1 mRNA to generate the transcriptionally active XBP1(S). This active form further promotes the expression of chaperone genes, which are essential for proper protein processing. Furthermore, as an adaptive response, the unfolded protein response (UPR) regulates transcription and translation by activating ER transmembrane proteins such as IRE1α, PERK, and ATF6 to alleviate ER stress. These mechanisms and pathways work synergistically to maintain ER homeostasis, protect cells from stress damage, and promote cell adaptability and survival. The retina converts light signals into electrical signals and is one of the most oxygen-intensive tissues. Diabetes, excessive light exposure, and ischemia and hypoxia can all lead to oxidative stress in the retina, accompanied by ER stress.

[0615] Studies have shown that light exposure can trigger stress responses in photoreceptor cells and retinal pigment epithelial cells (RPE cells) in vitro, including oxidative stress, endoplasmic reticulum stress, and autophagy. Endoplasmic reticulum stress has also been observed in the retina of light-damaged mice. Moreover, inhibiting endoplasmic reticulum stress helps reduce light-induced photoreceptor apoptosis, suggesting that endoplasmic reticulum stress may be an important target for treating retinal damage diseases. MSC-sIVs can inhibit endoplasmic reticulum stress signals, a finding that provides strong support for the use of MSC-sIVs in the treatment of diseases such as retinal oxidative stress and damage.

[0616] Example 13: MSC-sIVs promote proliferation, chemotaxis, and migration of human corneal epithelial cells

[0617] 1. Experimental methods

[0618] 1.1 Experimental Reagents

[0619] Table 18 Experimental reagents

[0620] 1.2 Culture of human corneal epithelial cells

[0621] Human corneal epithelial cells (HCEC) were provided by Wuhan Pronocell Life Science Co., Ltd. and are adherent, epithelial-like cells. The complete culture medium used was: DMEM / F12 (Invitrogen, USA) + 15% FBS (Invitrogen) + 5 μg / ml Insulin (Procell, PBI80332) + 10 μg / ml human EGF (Gibco, PHG0311) + 1% P / S. The cells were cultured in a 37°C, 5% CO2 incubator. Cryopreservation conditions: 55% basal medium + 40% FBS + 5% DMSO.

[0622] Cell recovery: Thaw cells in cryovials rapidly in a 37°C water bath, dilute with complete medium 1:10, centrifuge at 1000g for 5 min, resuspend cells, and transfer to a T25 culture flask containing 5 mL of complete medium.

[0623] Passaging steps: After aspirating the original culture medium each time, add PBS to rinse the cells and discard them. Add 1 mL of 0.25% trypsin solution (containing EDTA) to digest the cells. After the cells are digested under a microscope, add 3 mL of complete culture medium to stop digestion. Transfer and collect the cell suspension and centrifuge at 1000g for 5 minutes. After centrifugation, aspirate and discard the supernatant, resuspend in complete culture medium and transfer to a new culture flask. Change the culture medium every 2-3 days. When the cell fusion reaches 80%, subculture at a subculture ratio of 1:2-1:3, and use P3 to P5 cells for subsequent experiments.

[0624] 1.3 CCK-8 assay for cell proliferation

[0625] HCEC were inoculated into 96-well plates, starved and cultured in a 1-fold dilution of complete medium, and grown to 60% confluence. Different concentration gradients of sIVs or sEVs (prepared in Example 2) (2.5, 5, 10, 20, 40 μg / ml) were added. After 24 hours and 48 hours, the sample absorbance was measured at 450 nm using a microplate reader for 30 minutes using a CCK-8 kit. The relative absorbance of sIVs or sEVs (20, 40 μg / ml) was calculated using the absorbance of the control group at 450 nm for 24 hours and 48 hours as the baseline, and the proliferation effect curve of sIVs or sEVs at this concentration on HCEC cells was plotted.

[0626] 1.4 Transwell assay for HCEC chemotaxis after sIVs and sEVs

[0627] The Transwell chamber was activated with DF-12 basal medium containing 1% P / S, and 30 minutes later, HCEC was incubated with DF-12 basal medium containing 1% P / S. Different concentration gradients of sIVs or sEVs (2.5, 5, 10, 20, and 40 μg / ml) were added to the lower chamber of the experimental group. After incubation for 24 hours, cells attached to the upper surface of the filter membrane were removed. Migrated cells were fixed with 4% glutaraldehyde and stained with 0.5% crystal violet. Cells were counted under an inverted microscope. Cells in the photos were processed and counted using ImageJ.

[0628] 1.5 Scratch assay to detect HCEC migration after sIVs and sEVs

[0629] A 6-well plate was pre-marked, and HCECs were plated and grown to 80-90% confluence. After fixing with a ruler, a straight and uniform scratch was made on the bottom of the well plate using the tip of a sterile 1 mL pipette. Different concentration gradients of sIVs or sEVs (2.5, 5, 10, 20, 40 μg / ml) were added to the treatment group, and an equal volume of PBS was added to the control group. Wound closure was recorded using a fluorescence microscope at 0 h, 6 h, 12 h, and 24 h after the scratch and measured using ImageJ software. Wound closure ratio = [cell-free area (0 hour) - cell-free area (target hour)] / cell-free area (0 hour) × 100%.

[0630] 2. Experimental results

[0631] 2.1 MSC-sIVs promote HCEC proliferation in vitro

[0632] CCK-8 assay results showed that after 24 hours, all sIVs at different concentrations significantly promoted HCEC proliferation, while there was no significant difference between sEVs and the control group (Figure 78). The effect of sIVs on HCEC proliferation was significantly greater than that of sEVs after 24 hours (Figure 79). After 48 hours, all sIVs at different concentrations significantly promoted HCEC proliferation in a concentration-dependent manner. Only 40 μg / ml sEVs promoted HCEC proliferation, while the remaining sEV concentrations showed no significant difference from the control group (Figure 80). The effect of sIVs on HCEC proliferation was significantly greater than that of sEVs after 48 hours (Figure 81). We then applied sIVs and sEVs at the same effective concentrations (20 μg / ml and 40 μg / ml) to HCEC and dynamically monitored the proliferation of HECEs. 20 μg / ml sIVs significantly promoted HECE proliferation after both 24 and 48 hours, significantly more so than sEVs at the same concentration (Figure 82). 40 μg / ml sIVs significantly promoted the proliferation of HECE after 24 and 48 hours of treatment, which was more significant than the same concentration of sEVs ( Figure 83 ).

[0633] 2.2 MSC-sIVs promote HCEC chemotaxis in vitro

[0634] The results of the Transwell experiment showed that after 24 hours, sIVs and sEVs at concentrations of 5, 10, 20, and 40 μg / ml could promote the chemotaxis of HCEC in a concentration-dependent manner, and the number of cells passing through the chamber was significantly higher than that of the control group ( Figure 84 ). At the same concentration, the effect of sIVs was more significant than that of sEVs ( Figure 85 ).

[0635] 2.3 MSC-sIVs promote HCEC scratch repair in vitro

[0636] The results of the scratch assay showed that sIVs and sEVs could significantly promote the migration of HCEC ( FIG. 86 ). At 12 and 18 hours, the ability of sIVs to promote migration was more significant than that of sEVs ( FIG. 87 ).

[0637] 3. Summary

[0638] Compared with MSC-sEVs, MSC-sIVs can promote HCEC proliferation, chemotaxis, and scratch repair in vitro, effectively improving cell damage. It is expected that it can effectively improve corneal diseases caused by corneal damage and has the potential to repair corneal epithelial damage.

[0639] Example 14: MSC-sIVs promote the repair of corneal epithelial stroma in a mouse corneal trauma model

[0640] 1. Experimental methods

[0641] 1.1 Construction of corneal trauma model

[0642] Corneal trauma model: C57BL / 6 mice (6-8 weeks old) were housed under sterile conditions at the Laboratory Animal Center. Ofloxacin eye drops were administered three times daily the day before surgery. Mice were anesthetized via intraperitoneal injection and mydriasis was dilated with tropicamide compound eye drops. After topical anesthesia, excess moisture was removed with a cotton swab, and the cornea was fully exposed. A 2 mm trephine was used to mark the center of the cornea (below the limbus), and the entire corneal epithelium and anterior stroma were scratched using an AlgerBrush II corneal remover. Antibiotic gel was applied postoperatively.

[0643] 1.2 Affinity of corneal epithelium and stroma for sEVs and sIVs in corneal trauma model

[0644] MSC-derived sIVs and sEVs (prepared in Example 2) were labeled and stained with the membrane-labeling dye DiD. Mice undergoing corneal trauma were instilled with labeled sIVs and sEVs and fixed with 4% paraformaldehyde 4 hours later. The cells were embedded in OCT and stored at -80°C. Endocytosis was examined by laser confocal microscopy after cryosectioning.

[0645] 1.3 Treatment of corneal trauma in mice with the same concentration (0.1 μg / ml) of MSC-sIVs and MSC-sEVs

[0646] After establishing the corneal wound model, mice were divided into a PBS group, a 0.1 μg / μl sIVs treatment group, and a 0.1 μg / μl sEVs treatment group for treatment. 3.5 μl was administered twice a day.

[0647] 1.3 Fluorescein sodium staining shows the extent of corneal epithelial defect

[0648] Adjust the slit lamp biomicroscope to a clear field of view and position the mouse for optimal ocular surface observation. Mouse corneal epithelial fluorescein sodium staining: 4 μL of 0.1% sodium fluorescein solution was used for 10-15 seconds. Excess fluorescein sodium solution was removed with a cotton swab. Corneal epithelial defect area and corneal area were calculated using ImageJ. Corneal lesion closure ratio = corneal epithelial defect area (t hour) / corneal area (t hour) × 100%.

[0649] 2. Experimental results

[0650] 2.1 The absorption rate of MSC-sIVs at the corneal injury site is higher than that of MSC-sEVs

[0651] Equal amounts of DiD-labeled MSC-sIVs and MSC-sEVs were applied to the eyes. Four hours later, the eyeballs were harvested and frozen sections were taken. The results are shown in Figure 88. The middle column shows positive vesicle signals. After application, the vesicles were mostly distributed in the broken ends of the corneal injury. The corneal epithelial broken ends internalized MSC-sIVs more than MSC-sEVs. MSC-sIVs were distributed in the exposed corneal stroma, while MSC-sEVs hardly remained in the corneal stroma. This suggests that the damaged area of ​​the cornea has a higher absorption rate of MSC-sIVs, and MSC-sIVs can remain in the damaged area for a longer period of time, exerting their therapeutic effects, and have great application potential.

[0652] 2.2 MSC-sIVs significantly promoted the healing of corneal epithelial defects in mice

[0653] Equal amounts of MSC-sIVs and MSC-sEVs were used for eye drop treatment, and sodium fluorescein staining was performed before treatment and on the first and second days after treatment to show the corneal defect site. Representative slit lamp images and sodium fluorescein staining images are shown in Figure 89. The experimental results (Figure 90) showed that MSC-sIVs and MSC-sEVs significantly promoted corneal wound healing compared with the control group, and on the first day after treatment, the therapeutic effect of MSC-sIVs was more significant than that of MSC-sEVs.

[0654] Example 15: MSC-sIVs promote corneal sensitivity recovery after corneal injury

[0655] 1. Experimental methods

[0656] The corneal trauma model was constructed in the same manner as in Example 14. Corneal sensitivity was measured using a Cochet-Bonnet sensory meter (Luneau Ophtalmologie, Chartres Cedex, France). The tip of the fiber monofilament was used to contact the cornea. The length of the monofilament varied from 6.0 cm to 0.5 cm in increments of 0.5 cm until the corneal sensitivity threshold was found. The corneal sensitivity threshold was defined as the number of attempts exceeding 50% (at least 3 or more out of 5 attempts) resulting in stimulation of the corneal blink reflex. Once the length of the monofilament reached the threshold (maximum length 6 cm), the number of test attempts was increased from 5 to 10 to improve its accuracy.

[0657] 2. Experimental results

[0658] Corneal sensitivity threshold was detected by corneal perceptometer on the 7th and 14th days after treatment. The results showed (Figure 91) that MSC-sIVs could significantly promote the recovery of corneal sensitivity on the 7th day after treatment. On the 14th day after treatment, both MSC-sIVs and MSC-sEVs significantly promoted the recovery of corneal sensitivity, with the effect of MSC-sIVs being more significant.

[0659] 3. Summary

[0660] MSC-sIVs can quickly repair corneal epithelial damage and promote the recovery of corneal sensitivity. Its therapeutic effect is better than MSC-sEVs and it has great potential for clinical application.

[0661] Example 16: MSC-sIVs promote corneal sensitivity recovery after corneal alkali burn

[0662] 1. Experimental methods

[0663] 1.1 Construction of corneal alkali burn model

[0664] Corneal alkali burn model: C57BL / 6 mice (6-8 weeks old) were housed under sterile conditions at the Laboratory Animal Center. Ofloxacin eye drops were administered three times daily the day before surgery. Mice were anesthetized via intraperitoneal injection and mydriasis was dilated with tropicamide compound eye drops. After topical anesthesia, excess moisture was removed with a cotton swab, and the cornea was fully exposed. A 2 mm diameter filter paper was dipped in 1 M sodium hydroxide solution, and excess moisture was removed. The filter paper was placed in the center of the cornea for 1 minute, and the ocular surface was irrigated with saline for 1 minute. Antibiotic gel was applied postoperatively.

[0665] 1.2 Using a slit lamp to photograph corneal neovascularization

[0666] Slit lamp photographs of the side of the mouse cornea were taken before modeling, 3 days, 7 days, 10 days and 14 days after modeling, showing the blood vessels behind the corneal limbus and the new blood vessels growing into the cornea.

[0667] 1.3 Corneal flat mount showing corneal neovascularization

[0668] Fourteen days after modeling, mice were killed by cervical dislocation, and eyeballs were removed and fixed in PFA for 2 hours. Corneas were removed and fixed in PFA overnight at 4°C. Blocking solution was then added for 2 hours at room temperature. Primary antibody CD31 (R&D) was incubated overnight at 4°C, followed by washing of the primary antibody for 6 hours. Secondary antibody donkey anti-sheep IgG-594 (abcam) was incubated overnight at 4°C, followed by washing of the secondary antibody for 6 hours. Slides were then mounted with anti-fluorescence decay mounting medium. Images were taken using a confocal microscope.

[0669] 2. Experimental results

[0670] Figure 92 shows that starting on day 3, corneal vessels thickened in the alkali burn group. From day 7 to day 14, new corneal vessels emerged from the limbus and entered the cornea. On day 14 in the control group, new vessels were seen extending from the limbus to the center of the cornea. Figure 93 clearly shows the extent of new vessel invasion into the corneal tissue. Statistically, sIVs treatment significantly reduced the invasion of new vessels, with a superior effect compared to sEVs (Figure 94).

[0671] 3. Summary

[0672] MSC-sIVs can reduce corneal neovascularization, and its therapeutic effect is better than MSC-sEVs, which has great potential for clinical application.

[0673] In addition, according to the method described in the present invention, the inventors also prepared sIVs and sEVs using other adult stem cells such as rat bone marrow mesenchymal stem cells (BMSCs) and mouse adipose mesenchymal stem cells (ADSCs). Similar to umbilical cord mesenchymal stem cells, the particle size distribution range of sIVs from BMSCs and ADSCs is narrow and the particle size is small, while the particle size distribution range of sEVs is wide and the particle size is large, and the yield of sIVs is much higher than that of sEVs. Compared with sEVs, the retina has a stronger endocytosis of sIVs from BMSCs and ADSCs, and sIVs has a stronger protective effect on the retinal function of rd10 mice, and can more effectively reduce retinal cell apoptosis and reduce retinal rhodopsin loss. The preparation and testing experimental steps of sIVs and sEVs from BMSCs and ADSCs refer to the above examples and will not be repeated here.

[0674] In summary, the above Examples 1-3 demonstrate the collection process of sIVs and the parameter optimization process. Example 4 demonstrates the physical properties of sIVs from the perspectives of electron microscopic morphology, particle size, characteristic proteins and distribution within living cells, and confirms that sIVs has high thermal stability. Examples 5-7 demonstrate the uniqueness of sIVs and the significant differences between sIVs and sEVs from the perspectives of proteins, nucleic acids and lipids, respectively. Example 8 shows that cells cultured in vitro and retinal tissue in vivo have a higher absorption efficiency for sIVs. Examples 8 and 9 show that sIVs can load drugs such as small molecule drugs and protein drugs and deliver them to cells or tissues, and their drug loading rate, encapsulation rate and delivery effect are significantly better than sEVs based on exosomes. The above nine examples demonstrate that the in vitro collection method of sIVs described in the present invention is reliable, and the sIVs collected in the present invention are a unique type of intracellular vesicle population, which has a composition of biomacromolecules such as proteins, nucleic acids, and lipids that is different from the sEVs reported in the prior art. They also have excellent cell tissue affinity and the ability to carry and deliver drugs, and have very good application and research value in the medical field.

[0675] The above Examples 10-11 show that we evaluated the therapeutic effects of MSC-sIVs and MSC-sEVs in two mouse retinal damage models (retinal damage model caused by blue light irradiation and rd10 retinitis pigmentosa genetic model), and found that MSC-sIVs showed better efficacy in protecting retinal photoreceptor cells and had better therapeutic effects. Example 12 confirmed that MSC-sIVs exerts a protective effect on the retina by inhibiting endoplasmic reticulum stress signals. Examples 13-15 confirmed that MSC-sIVs can promote corneal epithelial cell growth in vitro, and in the corneal damage model, it can promote corneal epithelial and matrix repair. Example 16 confirmed that MSC-sIVs can inhibit pathological corneal neovascularization. In summary, sIVs produced by adult stem cells can treat ophthalmic diseases such as retinal degeneration and corneal damage, and its effect is better than sEVs. The intracellular nanovesicles derived from adult stem cells of the present invention have very good application and research value in the field of medicine.

[0676] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0677] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0678] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A method for preparing intracellular nanovesicles, comprising the following steps: (1) dispersing the cells in a suspension solvent and subjecting them to ultrasonic treatment; (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding cell membranes and organelle fragments, and taking the supernatant; (3) The supernatant obtained in step (2) is subjected to ultracentrifugation to obtain the precipitate as the intracellular nanovesicles.

2. The method according to claim 1, characterized in that The amplitude of the ultrasonic treatment in step (1) is 20%-25%, and / or the time of the ultrasonic treatment is 10-20s; Preferably, the amplitude of the ultrasonic treatment in step (1) is 20%; and / or the time of the ultrasonic treatment is 15 s, on 2 s, off 2 s.

3. The method according to claim 1, characterized in that The number of centrifugal treatments in step (2) is two, and the respective parameters are: 1000-3000g, 5-20 minutes; 10000-30000g, 20-40 minutes.

4. The method according to claim 1, characterized in that The parameters of the ultracentrifugation treatment in step (3) include 100000-180000g, 50-100 minutes.

5. The method according to claim 1, characterized in that One or more of the ultrasonic treatment, centrifugation treatment and ultracentrifugation treatment is performed at 0-5°C.

6. The method according to any one of claims 1 to 5, characterized in that: The cell is at least one of a stem cell, a cardiomyocyte, a human embryonic kidney cell, a macrophage, a T cell, and a tumor cell; Preferably, the stem cells are mesenchymal stem cells, and the mesenchymal stem cells are preferably selected from: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells.

7. A vesicle prepared according to the method of any one of claims 1 to 6.

8. Use of the vesicle prepared by the method according to any one of claims 1 to 6 as a drug carrier in the preparation of a drug for preventing and / or treating a disease, wherein the disease is preferably an eye disease.

9. A method for preparing a drug-loaded vesicle, comprising using the vesicle prepared by the method according to any one of claims 1 to 6 as a drug carrier for drug loading; Preferably, the drug delivery method is selected from: one or more combinations of genetic engineering, chemical synthesis, viral vector, ultrasound, electroporation, and co-incubation.

10. A drug-loaded vesicle, wherein the vesicle prepared by the method according to any one of claims 1 to 6 is used as a drug carrier; Preferably, the drug is rapamycin or interleukin-10.

11. Use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of drugs for preventing and / or treating retinal diseases, corneal diseases or repairing corneal damage.

12. The use according to claim 11, characterized in that The vesicles are prepared by a method comprising the following steps: (1) dispersing mesenchymal stem cells in a suspension solvent and subjecting them to ultrasonic treatment; (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding cell membranes and organelle fragments, and taking the supernatant; (3) The supernatant obtained in step (2) is subjected to ultracentrifugation to obtain the precipitate as the intracellular nanovesicles.

13. The use according to claim 12, characterized in that The amplitude of the ultrasonic treatment in step (1) is 20%-25%, and / or the time of the ultrasonic treatment is 10-20s; Preferably, the amplitude of the ultrasonic treatment in step (1) is 20%; and / or the time of the ultrasonic treatment is 15 s, on 2 s, off 2 s.

14. The method according to claim 12, characterized in that The number of centrifugation treatments in step (2) is two times, and the respective parameters are: 1000-3000g, 5-20 minutes; 10000-30000g, 20-40 minutes; and / or, The parameters of the ultracentrifugation treatment in step (3) are 100000-180000 g, 50-100 minutes.

15. The use according to claim 11, characterized in that The mesenchymal stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta and amniotic fluid, and amniotic membrane mesenchymal stem cells.

16. The use according to any one of claims 11 to 15, characterized in that: The retinal diseases are retinal damage and degeneration and related diseases; Preferably, the corneal disease is corneal neovascularization.

17. The use according to claim 11, characterized in that The retinal damage-related disease is selected from: glaucoma, diabetic retinopathy, ischemic retinopathy, and light-induced retinopathy; Preferably, the retinal degeneration-related disease is selected from: retinitis pigmentosa, age-related macular degeneration, Stargardt's disease, cone-rod dystrophy, congenital stationary night blindness, Leber congenital amaurosis, Best's vitelliform macular dystrophy, Central macular dystrophy, Bietti crystalline corneal retinal dystrophy, Usher's syndrome.

18. The use according to claim 11, characterized in that The retinal disease is selected from the group consisting of: photoretinopathy, diabetic retinopathy, ischemic retinopathy, and retinitis pigmentosa.

19. The use according to claim 11, characterized in that The drug is administered to the eye, preferably in the following ways: eye drops, eye ointment, subconjunctival injection, intravitreal injection, especially intravitreal injection and eye drops.

20. The use according to claim 11, characterized in that The medicament is a cream, foam, cream, ointment, emulsion, liquid solution, eye drop, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, especially an injection.

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