CN118207158B

CN118207158B - Preparation method and application of intracellular-source nano vesicles

Info

Publication number: CN118207158B
Application number: CN202410605857.XA
Authority: CN
Inventors: 张晓敏; 李筱荣; 张慧
Original assignee: TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
Current assignee: TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
Priority date: 2023-06-21
Filing date: 2024-05-16
Publication date: 2024-08-27
Anticipated expiration: 2044-05-16
Also published as: CN118207158A

Abstract

The invention discloses a preparation method and application of a nanometer vesicle from intracellular sources, in particular to application as a drug carrier. The method can efficiently enrich the nano vesicles from cells, and compared with small extracellular vesicles which are separated from cell supernatant and take exosomes as main components, the obtained intracellular nano vesicles have different lipid, protein and nucleic acid components, larger yield, smaller particle size and narrow particle size distribution range, are more stable at different temperatures, have higher encapsulation rate and drug loading rate when being used as carriers for drug loading, are easier to be absorbed by tissues when being administered in vivo, for example, the loaded drug can be absorbed by retina more quickly in the form of intravitreal cavity injection, and have very good application and research values in the medical field.

Description

Preparation method and application of intracellular-source nano vesicles

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of a nano vesicle from cells, and particularly relates to application of the nano vesicle as a drug carrier.

Background

In order to solve the problems of water solubility, biocompatibility, immunogenicity, toxic side effects, long action duration and the like of drugs, various bionic drug carriers are widely developed, such as chitosan, liposome, in-vitro reconstituted lipoprotein, biological vesicle and the like. The double-layer lipid membrane has better firmness and stability, and can be used as a drug carrier with wider administration route, higher drug-carrying stability, drug solubilization capacity and drug bioavailability. Biological vesicles have been reported for decades as drug carriers in injections, oral suspensions or emulsions, eye drops, nasal drops, and topical dosage forms.

Cell secretions, known as "extracellular vesicles" (Extracellular Vesicles, EV), released from cells into the blood circulation, either into the extracellular or plasma, have been of great interest and research. The "extracellular vesicles" are mainly classified into exosomes, microbubbles or microparticles and apoptotic bodies, wherein the main component is exosomes. Exosomes are vesicles formed by twice invagination of cell membranes, are secreted out of cells after substance exchange in the cells, and participate in intercellular information exchange and substance transport. It contains certain biomolecules specific to the parent cell and has various bioregulation functions. However, exosomes suffer from the disadvantages of difficult collection, exogenous contamination, and difficult storage, limiting their clinical transformation. According to the guidelines of minimal information on extracellular vesicle studies (MISEV) published in 2018, it is difficult to directly demonstrate that small particles isolated by conventional ultracentrifugation methods are purified exosomes. It is recommended to use terms such as Small extracellular vesicles (Small EVs, svvs) with a diameter of less than 200 nm. (thus, in the present invention, we use sEVs to represent small extracellular vesicles enriched by ultracentrifugation).

However, there are also many nanoscale vesicles within the cell, which are located between membrane-rich organelles and are responsible for intracellular material transport and secretion pathways. Intracellular vesicles (Intracellular Nanovesicles, IVs) are produced by a process known as vesicle budding and can originate from a variety of organelles including the endoplasmic reticulum, golgi apparatus, endosomes and plasma membranes. They consist of a variety of membrane-rich particles such as constitutive secretory vesicles, synaptic vesicles, COP-coated vesicles, golgi derived vesicles, clathrin-coated vesicles, transport vesicles between endoplasmic reticulum and golgi apparatus, and the like. These IVs are important components of the cellular transport system that can transport substances, including proteins, RNAs and lipids, between organelles, thereby coordinating overall cellular functions. Furthermore, in these IVs, secretory vesicles are involved in the secretion of specific proteins, hormones and other biomolecules by exocytosis. A subset of secretory vesicles, also known as synaptic vesicles, play an important role. Previous studies have focused mainly on signaling and modulation of IVs regulating substance transport, but to date there has been no study and report on how to efficiently enrich and utilize these intracellular vesicles.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a preparation method and application of a nanometer vesicle of an intracellular source, in particular to application as a drug carrier. The invention collects IVs by ultrasonic cell combined with ultracentrifugation, and because of its Small size characteristics, we use Small intracellular nanovesicles (Small IVs, sIVs) to represent ultracentrifuged enriched intracellular nanovesicles in the invention.

In a first aspect of the present invention, there is provided a method for preparing an intracellular-derived nanovesicle, comprising the steps of sequentially subjecting a cell to an ultrasonic treatment, a centrifugal treatment, and an ultracentrifugation treatment.

Specifically, the method comprises the steps of:

(1) Dispersing cells in a suspension solvent, and performing ultrasonic treatment;

(2) Centrifuging the liquid obtained in the step (1) for one or more times, discarding cell membranes and cell organelle fragments, and taking supernatant;

(3) Performing ultracentrifugation treatment on the supernatant obtained in the step (2), and taking the precipitate as intracellular nano vesicles;

optionally, (4) resuspending the pellet from step (3).

Specifically, the cells in step (1) are isolated cells obtained after culturing, digesting and washing (the possibility of obtaining extracellular vesicles by isolation can be excluded by discarding cell culture medium, digesting and washing, etc.).

In particular, the method may further comprise a cell digestion and counting step; in some embodiments of the invention, the cell digestion step comprises: the cultured cells were grown to 90% confluence, the cell culture medium was discarded, the cells were washed, the cells were digested with pancreatin, and then the cells were neutralized and washed.

Specifically, the cells have a cell density in the suspension solvent of 1-4X 10 ⁶/mL, e.g., 1X 10 ⁶/mL, 2X 10 ⁶/mL, 3X 10 ⁶/mL, 4X 10 ⁶/mL. In some embodiments of the invention, the cell density is 1X 10 ⁶/mL.

In particular, the suspension solvent is any suitable buffer for culturing cells, such as PBS, tris buffer, glycine buffer. In some embodiments of the invention, the solvent is PBS.

In particular, the cells are derived from mammals, particularly humans.

In particular, the cell is at least one of a eukaryotic cell, in particular a stem cell, a cardiomyocyte, an epithelial cell, a macrophage, a lymphocyte (e.g. T cell, B cell), a tumor cell, a fibroblast, a glial cell and a dendritic cell; more specifically, the stem cells are adult stem cells.

Specifically, the adult stem cells refer to undifferentiated cells existing in an already differentiated tissue, which are capable of self-renewal and of specializing in forming cells constituting the type of tissue, and under specific conditions, the adult stem cells either generate new stem cells or differentiate according to a certain procedure to form new functional cells, thereby maintaining the dynamic balance of growth and deterioration of tissues and organs, for example, mesenchymal Stem Cells (MSCs), hematopoietic Stem Cells (HSCs), neural Stem Cells (NSCs), etc., particularly mesenchymal stem cells.

In particular, the adult stem cells (in particular mesenchymal stem cells) are derived from mammals, in particular humans.

In particular, the Mesenchymal Stem Cells (MSCs), 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 mesenchymal stem cells.

In some embodiments of the invention, the cells are mesenchymal stem cells, in particular umbilical cord mesenchymal stem cells (UC-MSCs).

In some embodiments of the invention, the cell is an epithelial cell, such as 293T cell, 293/CHE-Fc cell, BHL-100 cell, BRL 3A cell, chang cell, CHO-K1 cell, clone 9 cell, CRFK cell, haK cell, HK-2 cell, HTC cell, IEC-6 cell, L2 cell, LO2 cell, vero-E6 cell, WI-38 cell, WISH cell, WS1 cell, etc.

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

In some embodiments of the invention, the cell is a tumor cell, 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, hea S3 cells, hep-2 cells, hep 3B cells, hep G2 cells, HL-60 cells, HP cells, HT-1080 cells, HT-29 cells, huT 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, CC97 cells, MONK-4 cells, MONK-92 cells, PAU 1, PAU 7 cells, XC 1, U7 cells, XCu-93 cells, U7 cells, U1-35 cells, U7 cells, U1-K-35 cells, and the like.

In some embodiments of the 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, and the like.

Specifically, the amplitude of the ultrasonic treatment in step (1) is 20% -25% (e.g., 20%, 22%, 24%, 25%); in some embodiments of the invention, the amplitude of the sonication is 20%.

Specifically, the time of the ultrasonic treatment in step (1) is 10 to 20s (e.g., 10, 12, 14, 15, 16, 18, 20), preferably 15s; in some embodiments of the invention, the time of the sonication is 15s, on 2s, off 2s.

In some embodiments of the invention, the centrifugation in step (2) is performed twice, with the respective parameters:

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

10000-30000g (e.g. 10000, 15000, 20000, 25000, 30000 g), 20-40 minutes (e.g. 20, 25, 28, 30, 32, 35, 40 minutes).

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

In one embodiment of the invention, the second centrifugation is carried out at 20000g for 30 minutes.

Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100000-180000g (e.g., 100000, 120000, 140000, 150000, 160000, 180000), 50-100 minutes (e.g., 50, 60, 65, 70, 75, 80, 90, 100 minutes).

In one embodiment of the invention, the ultracentrifugation is performed at 150000g for 70 minutes.

Specifically, the resuspension solvent in step (4) is any buffer suitable for culturing cells, such as PBS, tris buffer, glycine buffer. In some embodiments of the invention, the suspending solvent is PBS.

In particular, one or more of the sonication, centrifugation, ultracentrifugation treatments are operated at low temperatures, for example 0-5 ℃ (e.g. 0, 1, 2, 3, 4, 5 ℃); for example, the sonication, centrifugation, and ultracentrifugation are all performed at 4℃or on ice.

In some embodiments of the invention, the method comprises: taking cell suspension with the density of 1X 10 ⁶/mL, placing an ultrasonic probe in the center of the liquid level, and performing ultrasonic treatment with the ultrasonic amplitude parameter range of 20% and the time parameter range of 15s, on 2s and off 2 s; transferring the liquid into a centrifuge tube for centrifugation, wherein the centrifugation parameters are 2000g multiplied by 10min and 20000g multiplied by 30min, and collecting supernatant; the supernatant was transferred to an ultracentrifuge tube for centrifugation, with centrifugation parameters of 150000g×70min.

In a second aspect of the invention there is provided a vesicle prepared by the method of the first aspect.

Specifically, the vesicles are small intracellular nanovesicles (small Intracellular Nanovesicles, sIVs).

Specifically, the vesicle is in the shape of a double-layer horseshoe or teacup.

In particular, the vesicles have an average particle size of 50-100nm (e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nm), in particular 65-85nm.

Specifically, the vesicles express TMEM214 protein.

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

In particular, the vesicles are stable at temperatures ranging from-80 ℃ to 37 ℃ (e.g., -80, -50, -20, -10, 0, 5, 25, 37 ℃).

In a third aspect of the invention there is provided the use of a vesicle prepared by the method of the first aspect as a drug carrier loaded biomolecule in the manufacture of a medicament for the prevention and/or treatment of a disease.

In a preferred embodiment of the invention, the disease is an ocular disease.

Specifically, the vesicles as a drug carrier carry a pharmaceutically active ingredient having a prophylactic and/or therapeutic effect on the diseases (e.g., the above-mentioned ocular diseases); the pharmaceutical active ingredient comprises one or more of chemical drugs, biological drugs (such as proteins, polypeptides, oligonucleotide drugs), natural drugs, nano-drugs, radiopharmaceuticals, photothermal therapy and photodynamic therapy drugs.

In some embodiments of the invention, the chemical is used for ocular diseases, such as, but not limited to, aceclidine, acetazolamide, anecortave, actetadine, atropine, facoline, azelastine, bacitracin, benfuralol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carboline, cartalol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cromolyn acid, cycloprotide, cyclosporine, dapiprazole, demercalin, dexamethasone, diclofenac, dichlorsulfa, dipivefrine, dorzolamide, diethylphosphinothioyl-choline, emedastine, epinastine, epinephrine, erythromycin, ethoxybenzomine, mecamylin, fludrocortisone, flupirone, fomivirgin, neomycin B, ganciclovir, gatifloxacin, gentamicin, post-mandrine, hydrocortisone, idocin, idol, idocin, iodine indomethacin, isoflavones, ketorolac, ketotifen, latanoprost, levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medroxyprogesterone, methazolamide, metilolol, moxifloxacin, naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast peragatanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, prednisolone, procaine, ranibizumab, rimexolone, scopolamine, szomib, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, timolol, tobramycin, travoprost, triamcinolone, trifluoracetam, trimethoprim, topiramate, unoprostone, arabinoside, xylometazoline, doxycycline, minocycline, rapamycin, curcumin, artemisinin, sorafenib, methotrexate, and the like, or a combination thereof; particularly those that are fat-soluble.

In some embodiments of the invention, the chemical is used in tumors, such as, but 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, daunomycin, mitoxantrone, bleomycin, mitomycin, ixabepilone, tamoxifen, flutalamine gonadorelin analog, megestrol, prednisone, dexamethasone, methylprednisolone, thalidomide, interferon alpha, calcium folinate, sirolimus, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, ceridinib, crenolanib, crizotinib, dabrafenib, dacomitinib, danusertib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib, icotinib, imatinib, inipa, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, mo Tisha, lenatinib, nilatinib, niraparib, oprozomib, olaparib, pazopanib, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, ruxolitinib, secatinib, saridegib, sorafenib, sunitinib, tasocitinib, telatinib, tivantinib, tivozanib, tofacitinib, trametinib, vande, veliparib, or a combination thereof; particularly those that are fat-soluble.

In one embodiment of the invention, the pharmaceutically active ingredient is rapamycin.

In other embodiments of the invention, the pharmaceutically active ingredient is selected from: doxorubicin, paclitaxel, carmustine, curcumin.

In particular, the polypeptides and proteinaceous agents may include, but are not limited to, cytokines (e.g., interleukins (e.g., IL-1, IL-2, IL-6, IL-10, IL-11), colony stimulating factors (e.g., granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF)), interferons (e.g., alpha, beta, gamma interferon), growth factors, tumor necrosis factors (e.g., TNF-alpha and TNF-beta), transforming growth factor-beta family or chemokine family, etc.), human hemoglobin, clotting factors, vascular endothelial growth factor antibody antagonists, proteinaceous hormones (e.g., insulin, glucagon, calcitonin, hypothalamic hormone, pituitary hormone, or gastrointestinal hormone, etc.), antibodies (e.g., monoclonal antibodies (e.g., bevacizumab, ranibizumab, polyclonal antibodies, dimers, multimers, multispecific antibodies or antibodies), enzymes, brain-co-drugs (phenylalanine lyase, arginase, nuclease, superoxide dismutase, glycosamarase, or glycosaminidase).

In one embodiment of the invention, the pharmaceutically active ingredient is interleukin 10 (IL-10).

In other embodiments of the invention, the pharmaceutically active ingredient is selected from: bevacizumab, ranibizumab, aflibercept, buxizumab, and fariximab.

In particular, the medicament may be administered by any suitable means of administration, in particular intraocular, such as eye drops, eye ointments, subconjunctival injections, intravitreal injections, retrobulbar injections, in particular intravitreal injections.

In particular, the medicament may be formulated in any suitable formulation, such as, but not limited to, creams, foams, ointments, emulsions, liquid solutions, eye drops, injections, powder injections, gels, sprays, suspensions, microemulsions, eye masks or contact lenses, and the like, particularly injections.

In a fourth aspect of the invention there is provided a method of preparing a drug-loaded vesicle comprising loading a vesicle prepared by the method of the first aspect of the invention as a drug carrier.

Specifically, the preparation method of the drug-loaded vesicle can be one or a combination of a plurality of methods of genetic engineering method, chemical synthesis method, virus vector method, ultrasonic method, electroporation method and co-incubation method, in particular ultrasonic method.

Specifically, the drug loading step includes: mixing the vesicles with pharmaceutically active ingredients, incubating, sonicating, and then incubating;

Optionally, the free pharmaceutically active ingredient is removed (e.g. by ultrafiltration centrifugation).

In particular, the incubation temperature is 35-40 ℃ (e.g. 35, 36, 37, 38, 39, 40 ℃), in particular 37 ℃.

In particular, the incubation time before sonication is from 5 to 20 minutes (e.g., 5, 10, 15, 20 minutes), especially 10 minutes.

In particular, the incubation time after sonication is from 0.5 to 2 hours (e.g. 0.5, 0.8, 1, 1.2, 1.5, 2 hours), in particular 1 hour.

In particular, the amplitude of the ultrasonic treatment is 25% -35% (e.g. 25%, 28%, 30%, 35%), in particular 25%.

In particular, the time of the ultrasonic treatment is 60-180s (e.g., 60, 80, 100, 120, 180 s), particularly 60s.

In some embodiments of the invention, the sonication parameters include: 25% power, 30s pulse/30 s pause for 6 cycles.

In some embodiments of the invention, the ultrafiltration membrane parameter is 100kD.

In particular, the method for preparing the drug-loaded vesicles may further comprise the steps of the method for preparing vesicles according to the first aspect of the invention.

In a fifth aspect of the invention there is provided a drug-loaded vesicle comprising as a drug carrier a vesicle prepared by the method of the first aspect of the invention.

In particular, the drug in the drug-loaded vesicles is as described in the third aspect of the invention.

In particular, the drug loading rate of the drug-loaded vesicles may be 40% -90% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%).

In particular, the encapsulation efficiency of the drug-loaded vesicles may be 40% -90% (e.g. 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%).

In one embodiment of the invention, the active ingredient is rapamycin; in other embodiments of the invention, the pharmaceutically active ingredient is selected from: doxorubicin, paclitaxel, carmustine, curcumin; in one embodiment of the invention, the pharmaceutically active ingredient is interleukin 10 (IL-10); in other embodiments of the invention, the pharmaceutically active ingredient is selected from: bevacizumab, ranibizumab, aflibercept, buxizumab, and fariximab.

Specifically, the preparation method of the drug-loaded vesicle can be as described in the fourth aspect of the invention.

In particular, the pharmaceutically active ingredient of the drug-loaded vesicles may be as described in the third aspect of the invention.

In a sixth aspect of the invention there is provided a method of preventing and/or treating a disease comprising the step of administering to a subject in need thereof a vesicle prepared by the method of the first aspect of the invention (e.g. a vesicle according to the fourth aspect of the invention).

In particular, the disease is preferably an ocular disease, as described in the third aspect of the invention.

In particular, the subject is a mammal, particularly a human.

Specifically, the vesicles serve as a drug carrier to carry a pharmaceutically active ingredient, for example, a pharmaceutically active ingredient against the ocular diseases described above; the active pharmaceutical ingredients comprise one or more of chemical drugs, biological drugs, natural drugs, nano-drugs, radiopharmaceuticals, photothermal treatment and photodynamic treatment drugs; in one embodiment of the invention, the active ingredient is rapamycin; in other embodiments of the invention, the pharmaceutically active ingredient is selected from: doxorubicin, paclitaxel, carmustine, curcumin; in one embodiment of the invention, the pharmaceutically active ingredient is interleukin 10 (IL-10); in other embodiments of the invention, the pharmaceutically active ingredient is selected from: bevacizumab, ranibizumab, aflibercept, buxizumab, and fariximab.

In particular, the administration may be by any suitable mode of administration, in particular intraocular administration, such as eye drops, eye ointments, subconjunctival injections, intravitreal injections, in particular intravitreal injections.

The invention provides a method for collecting intracellular nano vesicles by ultrasonically breaking cells, which is simple in operation of separating vesicles and high in yield of the obtained vesicles. Compared with extracellular vesicles taking exosomes as main components, the obtained small intracellular nano vesicles have smaller particle size, narrow particle size distribution range and stability at different temperatures. The obtained intracellular nano vesicles have good tissue compatibility, compared with extracellular vesicles, the intracellular nano vesicles have wider distribution range and degree when being injected into tissues in situ, and have higher encapsulation rate and drug loading rate when being used as carriers for drug loading, for example, the drugs loaded by the intracellular nano vesicles in the form of intravitreal cavity injection can be absorbed by retina more quickly, and have very good application and research values in the medical field (especially in eye disease treatment).

Drawings

FIG. 1 is a schematic diagram showing the flow and steps of a method for producing intracellular nanovesicles. Wherein a shows a flow chart and B shows steps.

FIG. 2 shows the optimization of the parameters for the separation of nanovesicles within cells. Wherein, figures 2A-2B show protein production (figure 2A) and vesicle production (figure 2B) of sIVs obtained at different times of action at 20% ultrasound amplitude, respectively; at 20% ultrasound amplitude, the duration of action exceeded less than 10 seconds or more than 20 seconds, and the vesicle production suddenly decreased. FIGS. 2C-2D show protein production (FIG. 2C) and vesicle production (FIG. 2D) of sIVs obtained at 15s of sonication time, respectively, at different sonication amplitudes; at an ultrasound time of 15s, the ultrasound amplitude was higher than 25%, and the vesicle yield suddenly decreased. Fig. 2E shows a transmission electron micrograph of sIVs obtained at 20% ultrasound amplitude, at different times of action, scale bar: 100nm. Fig. 2F shows a transmission electron microscope image of sIVs obtained at 15s of ultrasound action time, at different ultrasound amplitudes, scale bar: 100nm.

FIG. 3 shows transmission electron micrographs of sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells, respectively. Wide field scale: 200nm; close-up scale bar: 200nm.

FIG. 4 shows the results of a nanoparticle size analysis showing the particle size distribution of MSCs (A), 293T (B), hela (C) cells and sEVs and sIVs thereof.

FIG. 5 shows statistical analysis results of MSCs, 293T, hela cells and their sEVs and sIVs particle sizesp＜0.01，P < 0.001 represents a significant difference between groups).

FIG. 6 shows statistical analysis results (D, E, F) of the total protein production and the numbers of sEVs and sIVs vesicles (A, B, C) of MSCs, 293T, hela cells at equal cell numbersp＜0.01、P < 0.001 andP < 0.0001 represents significant differences between groups).

FIG. 7 shows Coomassie brilliant blue staining results showing the protein distribution of cells of MSCs (A), 293T (B), hela (C) and sEVs and sIVs thereof.

FIG. 8 shows Western blot results demonstrating expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD 81) from MSCs (A), 293T (B), hela (C) cells.

FIG. 9 shows transmission electron micrographs of MSCs (A), 293T (B), hela (C) sEVs and sIVs at different temperatures. Scale bar: 200nm.

FIG. 10 shows the results of the nanoparticle size analysis of sEVs and sIVs of MSCs, 293T, hela cells at different temperatures (-80 ℃,4 ℃ and 37 ℃). Wherein FIG. 10A shows the particle size distribution, and FIG. 10B shows the results of statistical analysis of particle size [ ]p＜0.05，P < 0.01 represents a significant difference between groups).

FIG. 11 shows the results of a proteomic analysis revealing the different protein expression profile of sIVs versus sEVs for MSCs, 293T and Hela cells.

FIG. 12 shows the sIVs unique proteins co-expressed in MSCs (A), 293T (B) and Hela cells (C), in a top-to-bottom arrangement of abundance, displaying the top 50 proteins of each.

Fig. 13 shows the results of the super-resolution microscope and the total internal reflection fluorescent structure illumination microscope. Among them, fig. 13A shows the tri-SIM mode (showing cell membrane), which shows the presence of CD63 positive (green) regions on the cell membrane surface, while almost no TMEM214 positive (red) regions were observed. FIG. 13B shows WIDEFIELD-2DSM pattern (whole cells shown) demonstrating the simultaneous presence of CD 63-positive and TMEM 214-positive signals in whole cells. Fig. 13C shows a time-division screenshot of dynamic observation of living cells, green on the left side of CD 63-labeled intracellular late endosomes, sEVs and cell membranes, sEVs shown by the 0s arrow just released by the cell membranes to the outside of the cells, gradually moving away from the cell membranes for 6min to 14min, and TMEM214 on the right side of red, showing that sIVs is dispersed in the cells and not released to the outside of the cells.

FIG. 14 shows a Wen diagram showing total protein species in MSCs (A), 293T (B) and Hela (C) cells, sEVs and sIVs.

FIG. 15 shows the results of principal component analysis of total proteins identified in MSCs (A), 293T (B) and Hela (C) cells, sEVs and sIVs.

FIG. 16 is a heat map showing the presence of differentially expressed proteins between sEVs and sIVs of MSCs (A), 293T (B) and Hela (C) cells.

FIG. 17 is a volcanic chart showing the first five significant differentially expressed proteins between sEVs and sIVs of MSCs (A), 293T (B) and Hela (C) cells.

FIG. 18 is a heat map showing differential expression of exosome markers between sEVs and sIVs of MSCs (A), 293T (B) and Hela (C) cells.

FIG. 19 is a heat map showing differential expression of organelle markers between sEVs and sIVs of MSCs (A), 293T (B), and Hela (C) cells.

FIG. 20 is a heat map showing differential expression of Clathrin family proteins between sEVs and sIVs in MSCs (A), 293T (B) and Hela (C) cells.

FIG. 21 shows the enrichment analysis of cell components of sIVs co-expressed proteins from MSCs, 293T and Hela.

FIG. 22 shows biological process enrichment analysis of sIVs co-expressed proteins from MSCs, 293T and Hela.

FIG. 23 shows the relative RNA abundance in sEV and sIVs of MSCs, 293T and Hela cells (ns shows no statistical difference).

FIG. 24 shows the percentage of small non-coding RNAs from small RNA reads of sEVs and sIVs from MSCs (A), 293T (B) and Hela (C) cells. miRNA: micro-RNA; snoRNA: small nucleolus RNAs; snRNA: small nuclear RNA; tRNA: transferring RNA; rRNA: ribosomal RNA.

FIG. 25 shows a Wen diagram showing the species of miRNAs contained in sEVs and sIVs of MSCs (A), 293T (B) and Hela (C) cells.

Fig. 26 shows the results of principal component analysis of MSCs (a), 293T (B) and Hela (C) cell miRNA datasets.

Fig. 27 shows a matchgraph showing the first 10 high abundance mirnas in sEVs and sIVs of MSCs (a), 293T (B) and Hela (C) cells.

Figure 28 shows a heat map showing the presence of more differentially expressed mirnas between sEVs and sIVs in MSCs (a), 293T (B) and Hela (C) cells.

Fig. 29 shows a volcanic plot showing the first 5 mirnas differentially expressed between sEVs and sIVs in MSCs (a), 293T (B) and Hela (C) cells. The abscissa represents fold change in expression (log 2 fold difference) of mirnas between different samples or comparison combinations, and the ordinate represents the level of significance of the expression difference.

Enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs derived MSCs is shown in fig. 30. A shows the first 10 entries of GO analysis, biological process (Biological Process, BP), cellular component (Cellular Component, CC) and molecular function (Molecular Function, MF). B shows KEGG enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of MSCs.

Enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of 293T cell origin is shown in fig. 31. A shows the first 10 entries of GO analysis, biological process (Biological Process, BP), cellular component (Cellular Component, CC) and molecular function (Molecular Function, MF). B shows KEGG enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of 293T cells.

Enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of Hela cell origin is shown in fig. 32. A shows the first 10 entries of GO analysis, biological process (Biological Process, BP), cellular component (Cellular Component, CC) and molecular function (Molecular Function, MF). B shows KEGG enrichment analysis of miRNA candidate target genes differentially expressed in sEVs and sIVs of Hela cells.

FIG. 33 shows the types and ratios of metabolites contained in sEVs and sIVs of MSCs (A), 293T (B) and Hela cells (C).

FIG. 34 shows the results of principal component analysis of lipids contained in MSCs (A), 293T (B) and Hela cells (C) sEVs and sIVs.

FIG. 35 is a heat map showing the differential lipid types contained in MSCs (A), 293T (B) and Hela cells (C) sEVs and sIVs.

FIG. 36 shows a bar chart of the lipid profile of MSCs (A), 293T (B) and Hela (C) cells sIVs versus sEVs. The abscissa of the graph represents the relative percentage change of the group versus the content of each substance. If the relative percentage change of the content is zero, the content of the substance in the two groups is the same; the relative percent change in content is positive, indicating that the content of the substance in sIVs groups is higher; the percentage change in content relative to the amount is negative, indicating that the content of the material in group sEVs is higher. The ordinate of the lipidomic histogram represents the classification information of lipids.

FIG. 37 shows results of examining the ability of intracellular nanovesicles to endocytose by RPE cells cultured in vitro. Among them, figures 37A and B show that the di-labeled sEVs and sIVs were incubated with RPE cells for 3h, 12h, 24h, and 48h. Green is cytoskeleton, red is vesicle, DAPI staining shows blue as nucleus. Scale bar: 20 μm. FIG. 37C shows statistical results of DiD fluorescence intensity in cells [ ]p＜0.05、P < 0.01P < 0.001 represents a significant difference between groups).

FIG. 38 shows results of examination of the ability of intracellular nanovesicles to endocytose by HRMECs cells cultured in vitro. Among them, figures 38A and B show that the di-labeled sEVs and sIVs were incubated with HRMECs for 3h, 12h, 24h and 48h. Green is cytoskeleton, red is vesicle, DAPI staining shows blue as nucleus. Scale bar: 20 μm. FIG. 38C shows statistical results of intracellular DiD fluorescence intensities [ ]p＜0.05、P < 0.01P < 0.001 represents a significant difference between groups).

FIG. 39 shows the results of examining the ability of intracellular nanovesicles to endocytose the retina. Among them, fig. 39A shows the distribution of the di markers sEVs and sIVs on retinal sections by subconjunctival injections for 24h and 48 h. Fig. 39B shows the distribution of the di markers sEVs and sIVs on retinal sections by intravitreal injections for 8h and 24 h. DAPI staining showed nuclei. Scale bar: 20 μm. Figure 39C shows the statistics of the intensity of the DiD fluorescence in the retina of the subconjunctival injection group. FIG. 39D shows statistical results of DiD fluorescence intensity in retinas of the intravitreal injection group [ (]p＜0.05、p＜0.01、P < 0.001 andP < 0.0001 represents significant differences between groups).

FIG. 40 shows the results of drug loading performance investigation of intracellular nanovesicles. Wherein, fig. 40A shows transmission electron microscopy imaging of vesicle morphology in natural or ultrasound loading state. Fig. 40B shows the nanoparticle size analysis results of vesicles under natural or ultrasound loading conditions, scale bar: 200nm.

FIG. 41 shows the results of drug loading performance investigation of intracellular nanovesicles. In this regard, FIG. 41A shows the detection of the chromatographic peak of Rapa in Rapa sEV and Rapa sIVs by high performance liquid chromatography, characterized by a distinct large peak and a small peak. Fig. 41B shows the effect of sEVs and sIVs on Rapa encapsulation efficiency (n=6). FIG. 41C shows the effect of sEVs and sIVs on the drug delivery efficiency of RapaP < 0.001 represents a significant difference between groups).

FIG. 42 shows the results of drug loading performance investigation of intracellular nanovesicles. Wherein, fig. 42A shows the retinal Rapa content at different times after subconjunctival injections of Rapa, svvs-Rapa, svs-Rapa. FIG. 42B shows the content of retina Rapa at 24 hours after intravitreal injection of Rapa, sEVs-Rapa, sIVs-Rapap＜0.05、p＜0.01、P < 0.001 andP < 0.0001 represents significant differences between groups).

FIG. 43 shows the drug loading rate of sIVs packages of IL 10.

FIG. 44 shows the results of flow cytometry for detecting T cell proliferation.

FIG. 45 shows the statistical results of flow cytometry for detecting T cell proliferation.P < 0.01 represents a significant difference between groups.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

Various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: cell culture

1. Extraction and culture of Mesenchymal Stem Cells (MSC)

Human umbilical cord is provided by Beijing shellfish Biotechnology, inc., and umbilical cord obtained from normal pregnancy without complications after caesarean section is immediately placed in saline containing penicillin (100U/mL) and streptomycin (100 mg/mL) and then transported to the laboratory within 4 hours. After removal of residual blood and blood vessels, the obtained umbilical cord was cut into 1-3mm pieces and digested with 0.1% type II collagenase at 37℃for 1 hour. The suspension was then filtered through a 100 mesh screen to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco modified Eagle medium/nutrient mix F12 complete medium (DMEM, invitrogen, USA). The medium contained 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin (scientific, carlsbad, calif.). Cells were inoculated in T175 flasks and cultured in a 5% CO ₂ incubator at 37 ℃. The medium was changed every 3 days. When cell fusion reached 80%, passaging was performed at a 1:2 ratio of subcultures, and experiments were performed using cells of P3 to P5.

2. Culture of 293T cells

Cells were cultured using DMEM complete medium (DMEM, invitrogen, USA) with 5% fetal bovine serum, 1% penicillin-streptomycin (scientific, carlsbad, CA) and incubated at 37 ℃ and 5% carbon dioxide. The medium was changed every 2-3 days. Cells were used in various experiments at third to sixth generation, 70-80% confluence.

3. Culture of Hela cells

Example 2: preparation of cell-derived nanovesicles

The flow of the production method of the intracellular nano vesicles is shown in figure 1.

1. Cell digestion and enumeration

Cells were grown to 90% confluence, the cell supernatant was blotted, the cells were washed 2 times with PBS, the cells were digested with pancreatin, and after digestion, the cells were neutralized and washed 3 times with PBS. Cell counts were then performed and the cell numbers were adjusted to 1×10 ⁶/mL using PBS.

2. Sonicating cells

Cell suspension 2mL with the density of 1X 10 ⁶/mL is taken and added to the bottom of a 50 mL centrifuge tube, an ultrasonic probe is placed at the center of the liquid level, the ultrasonic amplitude parameter range is 20%, the time parameter range is 15s, on 2s and off 2s, and the centrifuge tube is placed on ice. The liquid was then transferred to a 2mL centrifuge tube for centrifugation.

3. Collection of nanovesicles within minicells

The centrifugation parameters were 2000 g.times.10 min,20000 g.times.30 min. The supernatant was then collected and transferred to an ultracentrifuge tube, the centrifugation parameters being 150000g×70min, all performed on ice. The pellet obtained was resuspended using PBS as small intracellular nanovesicles (small Intracellular Nanovesicles, sIVs).

4. Collection of small extracellular vesicles

FBS is a necessary condition for in vitro cell culture, but it contains a large number of extracellular vesicles of bovine origin and is also present in the complete cell culture medium containing FBS. To remove bovine extracellular vesicles, FBS was centrifuged at 110000 Xg overnight (about 12 hours) at 4 ℃. When the cell fusion reached 60%, the cells were cultured in complete medium containing 10% FBS from which exosomes were removed for 48 hours. Then, the supernatant was collected and the extracellular vesicles were separated by ultracentrifugation at 4 ℃. The specific steps include 300g×10min, 2000g×10min, 10000g×30min, and 110000g×70min twice. The pellet obtained was resuspended with PBS and is an exosome-based small extracellular vesicle (small Extracellular Vesicles, sEVs).

Example 3: optimization of separation parameters for intracellular nanovesicles

Taking MSCs cells as an example, referring to the procedure of example 2, the process of optimizing the separation parameters of intracellular nanovesicles is shown in fig. 2.

1. The nanometer granularity analyzer detects sIVs output difference

And (3) taking sIVs of PBS (phosphate buffered saline) for resuspension, diluting to 1ml with PBS, detecting by using nanoparticle tracking analysis software (Nanoparticle TRACKING ANALYSIS, NTA) NTA 3.3 Dev Build 3.3.104, setting the temperature to 25 ℃, setting the laser to Blue488, setting the flow rate to 50, automatically detecting in a Mode of three times of sample injection, analyzing three times, and taking the peak value average value as a Mode particle size result. The camera mode is sCMOS. The laser type was Blue488 and the viscosity was 0.9cP.

2. Morphology of different cells sIVs by transmission electron microscopy

Taking sIVs after centrifugation, re-suspending in 200 mu L of PBS solution, uniformly mixing, and taking 10 mu L of sIVs solution and 4% PFA according to the volume ratio of 1:1, dripping the mixture on a clean plastic film to form liquid drops, then buckling the front surface of an electron microscope carbon net on the liquid drops, placing for 20min, carrying out negative dyeing on 10 mu L of phosphotungstic acid for 90s, baking the carbon net, and observing by using a HitacW-7500 transmission electron microscope.

FIGS. 2A and 2B show the protein yield and vesicle yield of sIVs obtained according to this implementation procedure, respectively, at an ultrasound amplitude of 20% with the selection of the action times of 5s, 10s, 15s, 20s, 25s, 30s and 60 s. The results showed a time of action below 10 seconds or exceeding 20 seconds, and a sudden drop in the number of vesicles obtained and in the protein yield. FIGS. 2C and 2D show the protein yield and vesicle yield of sIVs obtained according to this implementation procedure, respectively, with amplitudes of 20%, 25%, 30%, 35% and 40% chosen at an ultrasound exposure time of 15 s. The results show that at an ultrasound time of 15s, the ultrasound amplitude was higher than 25% and the vesicle yield was suddenly reduced. Fig. 2E shows transmission electron microscopy images of sIVs obtained according to this implementation procedure, with ultrasound amplitudes of 20%, 25%, 30%, 35% and 40% selected at an ultrasound time of 15 s. FIG. 2F shows a transmission electron micrograph of sIVs obtained according to this implementation procedure at 20% ultrasound amplitude, with ultrasound times of 5s, 10s, 15s, 20s, 25s, 30s and 60s selected.

The present optimization procedure showed that at 20% ultrasound amplitude, the duration of action exceeded less than 10 seconds or 20 seconds, and the vesicle production suddenly decreased. At an ultrasound time of 15s, the ultrasound amplitude was higher than 25%, and the vesicle yield suddenly decreased. An amplitude of 20% and an ultrasound time of 15s are the optimal parameters for collecting intracellular nanovesicles.

Example 4: sIVs has unique physical characteristics and high thermal stability

1. Laboratory instrument and materials

1.1 Experimental reagent

TABLE 1 Experimental reagents

1.2 Laboratory apparatus

Table 2 laboratory apparatus

2. Experimental method

2.1, Morphology of Transmission Electron Microscopy (TEM) observations sEVs and sIVs

Centrifuged sEVs and sIVs (prepared in example 2) were mixed by re-suspending in 200. Mu.L PBS solution, and 10. Mu.L sEVs and sIVs solution and 4% PFA were mixed in a volume ratio of 1:1, dripping the mixture on a clean plastic film to form liquid drops, then buckling the front surface of a carbon net on the liquid drops, placing the mixture for 20min, carrying out negative dyeing on 10 mu L of phosphotungstic acid for 90s, baking the carbon net, and observing the carbon net by using a HitacW-7500 transmission electron microscope.

2.2, Particle diameter of nanoparticle analysis detection sEVs and sIVs

And (3) taking PBS resuspended sEVs and sIVs, diluting to 1ml with PBS, detecting by using nanoparticle tracking analysis software (Nanoparticle TRACKING ANALYSIS, NTA) 3.3 Dev Build 3.3.104, setting the temperature to 25 ℃, setting the laser to Blue488, setting the flow rate to 50, setting the Mode to automatic detection, injecting three times, analyzing three times, and taking the peak value average value as the particle size result of Mode. The camera mode is set to sCMOS. The laser type was set to Blue488 and the viscosity was set to 0.9cP.

2.3 Coomassie Brilliant blue staining analysis sEVs and sIVs protein composition

After quantitative analysis by BCA, an equal amount of protein sample was taken, PBS was added to a volume of 20. Mu.l, 5. Mu.l of protein loading buffer (5X) was added, and the mixture was heated at 95℃for 5 minutes. The protein electrophoresis conditions were 100V,90min. After electrophoresis, coomassie brilliant blue ultrafast staining solution was added, and incubated at room temperature for 2 hours, and the solution was washed with pure water until the water became clear. The glue was photographed.

2.4 Exosome marker protein constructs of Western Blot analysis sIVs and sEVs

After protein detection by BCA kit, equal amounts of each group of protein samples were taken, PBS was added to volume to equal volume, protein loading buffer (5×) was added, and heated at 95 ℃ for five minutes. The gel was prepared according to SDS-PAGE kit instructions and an electrophoresis comb was inserted. Rest and wait for solidification at room temperature for 25 minutes. The prepared SDS gel was placed in a previously prepared electrophoresis tank. The electrophoresis comb was removed and the denatured proteins were injected into the loading well of SDS-PAGE. 1-4. Mu.l of marker was added on each side. And (3) adjusting the voltage to 60V, changing the voltage to 100V after the upper layer glue runs out, and stopping electrophoresis until the lowest bromophenol blue indication line is 1-2cm away from the bottom of the glass plate. The activated PVDF membrane was placed on the SDS-PAGE surface, and then filter paper and foam pads were placed on both sides of the gel and membrane, respectively. The column was gently rolled to remove air bubbles in the system and clamp the electrical clamp. And setting a black electrode as SDS-PAGE, a red electrode as PVDF film, transferring the film under constant pressure, and simultaneously placing an ice bag in an electrotransfer tank for cooling. After the completion of the electrotransfer, the PVDF membrane was put into a TBST solution containing 5% skimmed milk or 1% BSA, and blocked at room temperature for 2 hours. After the completion of the blocking, primary antibodies were added and incubated overnight on a refrigerator shaker at 4 ℃. The next day, the primary antibody was removed and the PVDF membrane was washed by adding TBST solution. Then, secondary antibody was added and incubated on a room temperature shaker for 2 hours. After the incubation was completed, the secondary antibody was removed again, and the PVDF membrane was washed 3 times with TBST solution for 10 minutes each. Finally, developing the PVDF film by using ECL hypersensitive luminescence liquid.

3. Statistical treatment

Experimental data were expressed as mean ± standard deviation @) And (3) representing. The experimental data were checked normally. All quantitative data were analyzed using SPSS 22.0. Analysis of variance was performed using One-way ANOVA and post hoc testing was performed using Least Significant Difference (LSD) analysis. For non-normal distribution data and data with variance non-uniformity, a non-parametric test was used, with P values <0.05 being statistically significant differences.

4. Experimental results

4.1 Morphology of transmission electron microscope displays sEVs and sIVs

SIVs of MSCs, 293T and Hela cells were enriched, respectively, and examined using transmission electron microscopy. As a result, as shown in FIG. 3, sEVs of the three cells had a round or horseshoe shape with a diameter of 100-200nm, a large number of sIVs, a round morphology and a diameter of less than 100nm. The electron microscopy results showed sIVs a diameter significantly less than sEVs a.

4.2 Nanoparticle tracking analysis showed sEVs and sIVs particle size distributions

As shown in FIG. 4, the particle size distribution of sEVs of MSCs, 293T cells and Hela cells is wide, the particle size is larger, the particle size distribution of sIVs is narrow, and the particle size is smaller.

As a result of statistical analysis, the average particle size of sEVs of MSC is 123.1+ -4.457 nm, and the average particle size of sIVs is 75.28 + -9.067 nm; the sEVs average grain diameter of 293T is 121.6+ -6.855 nm, and the average grain diameter of sIVs is 80.27+ -8.031 nm; the sEVs average particle size of Hela cells is 139.0+ -6.352 nm, and the sIVs average particle size is 68.53 + -10.04 nm; the sIVs particle size of all three cells is smaller than sEVs.

4.3 Comparison of yields sEVs and sIVs at equal cell amounts

To compare the yields of both types of vesicles, we collected sEVs and sIVs from both cell culture supernatant and adherent cells. Upon NTA detection, the results showed that the number of sIVs vesicles produced by 1×10 ⁷ cells was 10-to 20-fold higher than sEVs (fig. 6A, 6B, 6C), and the protein product in sIVs derived from 1×10 ⁷ cells was 20-to 40-fold higher than sEVs (fig. 6D, 6E, 6F). This indicates that sIVs yields well above sEVs.

4.4 SEVs and sIVs protein species distribution differences

Whole proteins of cells, sEVs and sIVs were separated by SDS-PAGE and total protein distribution was shown using coomassie blue staining. The staining result shows that the protein contained in the cells shows the most abundant bands and has a plurality of high-abundance protein bands; sEVs contains fewer protein types, and the high-abundance proteins are positioned at about 200kD and 70 kD; sIVs contains more protein species than sEVs, and high abundance proteins are located around 250kD and 55kD (fig. 7). The protein distribution was different from cell to cell, which initially indicated that sIVs and sEVs had different protein compositions and were different from the total protein distribution of both cells and sEVs. I.e., sIVs has a unique protein profile.

To further analyze the protein expression profile of the cells and their sEVs and sIVs. The expression of exosome marker proteins, alix, HSP70, TSG101, CD63, CD81, of cells and sEVs and sIVs under the protein conditions of Western blot detection and the like is used. The results are shown in FIG. 8. The cell sEVs expressed the most exosome marker protein, indicating that sEVs obtained in this example is an exosome-based extracellular vesicle. The cells themselves also express an amount of Alix, HSP70, TSG101, CD63; however, sIVs had substantially no expression of Alix and CD81, and the expression levels of HSP70, TSG101 and CD63 were also much lower than those of cells and sEVs, further indicating that sIVs did not feature exosomes, not precursors of exosomes in cells, nor debris from cell homogenates, sIVs was a unique intracellular vesicle population.

4.5 Comparison of stability of sEVs and sIVs at different temperatures

To evaluate the stability of the two vesicles at different temperatures, sIVs and sEVs suspensions from the three types of cells were split into three parts, respectively, and stored at different temperatures (-80 ℃, 4 ℃ and 37 ℃). After 24 hours, the morphology, size and amount of protein were assessed sEVs and sIVs. Both vesicles from three cells were stable at-80 ℃ and 4 ℃. However, the transmission electron microscopy image showed that the morphology of sEVs was impaired at 37 ℃, had irregular shapes, broken vesicles and rough boundaries (fig. 9), and the number of sEVs was also reduced (fig. 10B), while the morphology and particle number of sIVs remained stable at 37 ℃. The above results indicate that sIVs has higher thermal stability than sEVs.

4.6 Super-resolution imaging shows sIVs that the distribution in cells is different from sEVs

To gain insight into the intracellular distribution of sIVs, we identified by proteomic analysis that sIVs was a uniquely expressed protein compared to sEVs, i.e. the IV signature protein, which carried the green fluorescent protein and was imaged intracellularly in order to visualize the morphology of sIVs in the cell.

Proteins uniquely expressed in each cell type sIVs were identified by proteomic analysis of MSCs,293t, sIVs and sEVs of hela cells. By looking for intersections of these three uniquely expressed proteins through wien's diagram we found a total of 106 commonly uniquely expressed proteins (fig. 11). Subsequently, we ranked the expression abundance of these 106 proteins from high to low in MSCs,293T and Hela cells and showed the first 50 proteins (fig. 12A, 12B, 12C). We observed that TMEM214 protein was expressed most abundantly in MSCs and TMEM214 was expressed more abundantly in the other two cell types. TMEM214 is a transmembrane protein （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.）. involved in cellular processes such as vesicle transport and protein transport, and therefore, we used green fluorescent protein GFP to label TMEM214 to visually display sIVs in the intracellular state, while using GFP-labeled CD63 as a marker in the cell sEVs.

Super-resolution microscopy and total internal reflection fluorescent structure illumination microscopy （Total Internal Reflection Fluorescence Microscopy combined with Structured Illumination Microscopy , TIRF-SIM） showed that CD63 was visible on the cell membrane (fig. 13A, green), and TMEM214 was not expressed on the cell membrane (fig. 13A, red). This finding precludes sIVs from the possibility of cell membrane reconstitution and confirms that the origin and locus of activity of sIVs is located within the cell. Super-resolution microscopy using WILDFIELD-2DSM scan mode provided an overview of whole cell protein expression, and images showed significant expression of both CD63 and TMEM214 in the cell (fig. 13B). Subsequently, we observed dynamic changes in the structure of these protein markers in living cells under wide field conditions. And photographing every 10 seconds, and continuously photographing for 15 minutes to form a dynamic video. In the video screenshot, the process of sEVs dynamic release from the cell membrane to the outside of the cell was observed (fig. 13C, green, white arrow), whereas TMEM214 labeled sIVs was dispersed inside the cell and not released to the outside of the cell (fig. 13C, red). Visual microscopic imaging shows that sIVs is in a cloud-like dispersion distribution in cells and is not released outside the cells.

5. Knot (S)

In this example we take three cells, MSCs, 293T and Hela cells as examples, sIVs and sEVs were collected and characterized. The sIVs size is found to be significantly smaller than sEVs by transmission electron microscopy and nano-particle size analysis; the total protein expression patterns of cells sEVs and sIVs are different, and sIVs expresses exosome marker proteins in a low mode; at equal cell numbers, sIVs yields were significantly greater than sEVs. At-80 ℃, sIVs and sEVs are equivalent in stability, but at 37 ℃, sIVs is significantly better than sEVs in stability. sEVs was observed to be released outside the cell via the cell membrane by super-resolution imaging, while sIVs was subject to frequent activity inside the cell. In general, different cells can be collected by the method of the invention to give a population of identical sIVs vesicles containing unique protein constructs with higher stability at physiological temperatures and much higher yields than extracellular vesicles.

Example 5: quantitative proteomic analysis revealed sIVs to have a unique protein expression profile

1. Laboratory instrument and materials

1.1 Experimental reagent

TABLE 3 Experimental reagents

1.2, Laboratory apparatus

Table 4 Experimental apparatus

2. Experimental method

2.1 Preparation of protein Mass Spectrometry samples of cells and sEVs and slVs thereof

2.1.1 Protein extraction

1) To the cells, sEVs, sIVs (prepared in example 2) were added 220. Mu.l of urea lysate (8M urea, 50mM NH ₄HCO₃, protease inhibitor) respectively, and lysed at room temperature for 5 minutes.

2) ON-ice ultrasonication (energy 35%, ON 3S, OFF 3S, ultrasonic time total 2 min), inserting the sample tube into ice box, centrifuging at 20deg.C for 10min at 14,000g, collecting supernatant, and centrifuging again.

3) The BCA method detects protein concentration, and 100 μg of protein was taken per cell, svs.

2.1.2 Protein denaturation

1) DTT was added to each of the sample tubes of cells, sEVs and sIVs to a final concentration of 10mm and incubated at 37 ℃ for 1 hour to reduce the protein.

2) IAA was added to each of the sample tubes of cells, sEVs and sIVs to a final concentration of 40mM and incubated at room temperature for 1 hour in the absence of light.

3) The above sample numbers were first marked on the collection tube, the 10kDa ultrafiltration tube was equilibrated 2 times with HPLC grade methanol, each time methanol was 150. Mu.l in volume, 14,000g for 5min, 300. Mu.l of 50mM NH ₄HCO₃ was added, followed by washing twice, 100. Mu.g of the protein sample after reductive alkylation was added, centrifugation was performed at 14000g for 20min at 4℃and 300. Mu.l of 50mM NH ₄HCO₃ was added for three washing times, the new collection tube was replaced, and 75. Mu.l of 50mM NH ₄HCO₃ was added to the ultrafiltration tube.

2.1.3 Protease cleavage

1) 3 Μg of pancreatin for mass spectrometry was added and incubated in a 37℃incubator for 14-16 hours.

2) The next day, centrifugation was carried out at 4℃for 20 minutes at 14,000g, a volume of 50. Mu.l of 50mM NH ₄HCO₃, rinsing 2 times, and 1% (volume) formic acid was added to the collection tube to terminate the cleavage, and evaporated to dryness in vacuo at 60 ℃.

2.1.4 Pool fractionation

1) Mu.l of 0.1% aqueous formic acid was used to resuspend the samples and the concentration was measured using nanodrop. Next, about 10. Mu.g of peptide fragments were removed from each sample, and they were pooled into one sample S.

2) 6 Μg was removed from sample S for DDA mode mass spectrometry, while the remaining S sample was subjected to fractionation on a home-made high pH reverse phase column.

3) For fractionation, the required reagents were formulated as in table 5. Buffer a was 100% Acetonitrile (ACN) and Buffer B was 0.1% trifluoroacetic acid (TFA).

TABLE 5 fractionation reagent ratios

4) A layer of C18 film was loaded into a 200 microliter gun head using iron wire. Then, 30 mg of the high pH resistant C18 packing was dissolved with 200. Mu.l of Buffer A and the packing was fed to the fractionating column. Then, the mixture was centrifuged at 3,000g for 2 minutes at 4℃and washed once with 200. Mu.l of Buffer A and three more times with 200. Mu.l of Buffer B. Finally, the column is left for use.

5) The digested peptide was loaded onto a fractionation column and repeated 5 times.

6) The column was washed 3 times with 200. Mu.l Buffer B. Next, 150. Mu.l of eluents of different concentrations were added and elution was performed in a gradient. The 6% and 35% fractions were combined into one fraction and 1.5. Mu.g of peptide fragment was removed from each fraction for DDA mode mass spectrometry.

2.2 Cells and sEVs, sIVs liquid phase Mass Spectrometry parameters thereof

The digested peptide was loaded onto a home-made Trap column (100 micron x2 cm, C18 packing, particle size 3 microns, 120A) using phase a (0.1% formic acid, 2% acetonitrile and 97.9% water) at a flow rate of 3 μl/min. Subsequently, the Trap column was eluted with a different gradient of phase B (containing 97.9% acetonitrile, 2% water and 0.1% formic acid). These eluted peptide fragments were passed through an analytical column (150 μm x 15 cm, C18 packing, particle size 1.9 μm, 120A) to form a charged spray, and finally passed into a mass spectrometer detector.

The gradient of phase B was set as follows: 0 to 5%,2 to 10%,65 to 22%,91 to 35%,92 to 80%,105 to 80%,106 to 5%,120 to 5%, the flow rate of the whole process being maintained at 500 nanoliters/minute.

In performing DDA scans, mass spectrometry parameters were set as follows: the accumulation time of TOF MS is 0.25 seconds, the mass scanning range covers 300-1500 daltons (Da), only +2 to +5 valence ions are detected, and the mass deviation is required to be less than 50ppm. A maximum of 60 ions are monitored per cycle, and after each detection, the detected ions are isolated for 16 seconds. The fragmentation energy mode employs a dynamic fragmentation mode. The accumulation time of the Product is 0.04 seconds, and a high sensitivity scanning mode is adopted.

In the case of DIA scanning, the mass spectrum parameters are different: the accumulation time of TOF MS is 0.05 seconds, and the second-stage scanning adopts a high-sensitivity mode. The number of variable windows is set to 100, the accumulation time of each window is 30 milliseconds, and the mass scanning range is 300-1500Da. The SWATH Variable Window Calculator _v1.1 program was used to calculate the specific mass range for each variable window.

2.3 Cells and sEVs, sIVs protein mass spectrometry data processing and bioinformatics analysis thereof

Database searches were performed on raw data collected in DDA mode using Proteinpilot software (version 5.0.1) with trypsin as the cleavage mode. The database selected was the Uniprot human database, which contained 20431 annotated proteins, published in 2019, 7. Screening conditions were unused protScore to greater than 0.05. Proteinpilot search results were imported into SWATH software (version 2.0) as a database to quantify the data collected in DIA mode.

During the quantification process, 6 peptide fragments were selected per protein, and 6 transitions (ion pairs) were selected per peptide fragment. The confidence of the peptide fragment was set to 99% and the FDR (false positive rate) was set to 1%. Meanwhile, the modified peptide is removed, the peak extraction window is set to 10 minutes, and the quality deviation is controlled within 50 ppm. 2 endogenous peptide fragments were selected every 10 minutes to correct the retention time and the peak area output was taken as a quantitative value.

Processing of protein expression data involves the steps of: the original quantitative values were first log2 transformed to meet normal distribution and then normalized using the normal. After removing proteins without gene names, using R language stats package for differential analysis, protein with P value less than 0.05 and change multiple more than 1.5 times is selected as differential protein. Meanwhile, the corrected p value is set to be less than 0.05.

GO and pathway analysis is then performed through Cytoscape plug-in clueGo. In GO enrichment analysis, cellular Components (CCs), molecular Functions (MF) and Biological Processes (BP) were selected for analysis. Whereas in the pathway enrichment analysis databases kegg and reactome were selected for use.

Heatmap Principal Component Analysis (PCA) scores, venn plots, volcanic plots, etc. were plotted using the R language or Hiplot software. The kyoto gene and genome encyclopedia (KEGG) pathway enrichment analysis uses METASCAPE online analysis software.

2.4 ELISA experiments

MSCs sEVs and sIVs were collected, adjusted to equal mass, and operated according to the instructions of the enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) kit. Adding the sample into the hole coated with the antibody, incubating for 2 hours, and cleaning the hole plate; biotin is added, incubated for 1 hour, and the pore plate is cleaned; HRP was added and incubated for 1 hour, and the well plate was washed; adding TMB substrate, and incubating for 20 minutes; STOP solution was added until the color was apparent. Absorbance values were read for each well in the well plate using a microplate reader at a wavelength of 450nm/540nm.

3. Statistical treatment

Experimental data were expressed as mean ± standard deviation @) And (3) representing. The experimental data were checked normally. All quantitative data were analyzed using SPSS 22.0, and statistical analysis was performed between the two groups using t test. P values <0.05 are statistically significant differences.

4. Experimental results

4.1 Wen diagram shows the difference in the composition of cells and sEVs and sIVs proteins thereof

To characterize the molecular composition of sEVs and sIVs, we performed proteomic analysis of sEVs and sIVs derived from MSCs, 293T cells and Hela cells using label-free mass spectrometry techniques and performed a comparison analysis 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 of the three cells, and 2066, 2403, and 2272 proteins were found in sIVs of the three cells, respectively (fig. 14). There is some overlap in the protein species between them, but not exactly the same. The protein content in sIVs was more varied than in sEVs in all three cell types.

4.2 Principal component analysis shows that cells and sEVs and sIVs protein component differences

The protein composition of cells and their svvs, svs was analyzed by PCA (fig. 15), and the results indicated that cells, sEVs and sIVs exhibited different protein distribution patterns. sEVs and sIVs showed significant differences in protein expression among three different cell types. This suggests sIVs, unlike sEVs, has unique protein expression characteristics.

4.3 Quantitative protein differential analysis of cells and sEVs, sIVs

Further statistics indicate 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, 753 of which sIVs was significantly down-regulated compared to sEVs, 672 of which was significantly up-regulated, 468 of which the fold difference was greater than 10-fold, 227 of which sivs were significantly down-regulated compared to sEVs, 241 of which were significantly up-regulated (fig. 16A); 1841 differentially expressed proteins were found in 293T cells, of which sIVs was significantly down-regulated 780 compared to sEVs, 1061 was significantly up-regulated, of which the fold difference was greater than 10-fold 321, sIVs was significantly down-regulated 159 compared to sEVs (fig. 16B); there were 1474 differentially expressed proteins in Hela cells, 594 of which sIVs was significantly down-regulated compared to sEVs, 880 of which was significantly up-regulated, 341 of which was more than a factor of 10 fold difference, and 198 of which sIVs was significantly down-regulated compared to 143 of which was sEVs (fig. 16C). Further embodying the uniqueness of sIVs.

The proteins whose up-and down-regulation was most pronounced in sEVs and sIVs were not identical in the three cell types (fig. 17). However, the expression level of membrane-associated proteins was lower in sIVs, whereas endoplasmic reticulum and ribose-associated proteins were more abundant in sIVs. This preliminary suggests sIVs is a unique entity produced by the cell, not a precursor or fragment of a cell lysate or extracellular vesicle.

4.4 Cells and expression differences in exosome markers of sEVs, sIVs

We extracted MISEV2018 the list （Théry C., Witwer K. W., 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.）, of recommended exosome markers and compared sEVs and sIVs for the three cell types with these markers. sEVs showed higher expression levels of the exosome marker, while sIVs showed lower expression levels for most exosome marker proteins (fig. 18). This further illustrates that sIVs does not have exosome characteristics, sIVs is a unique vesicle from the interior of the cell.

4.5 Cells and differences in the expression of organelle marker proteins of sEVs, sIVs

We also performed comparative analysis of the organelle protein expression profiles of sEVs and sIVs in three cell types. Overall, sIVs contained intracellular organelle proteins at levels higher than sEVs (fig. 19). In particular sIVs showed elevated protein expression levels associated with membrane-enriched organelles such as endosomes, endoplasmic reticulum, and golgi apparatus. In contrast sEVs contained a much richer cell membrane protein (fig. 19). This indicates sIVs has intracellular characteristics.

4.6 Cells and differences in the expression of Clathrin protein families of sEVs, sIVs

Among the proteins contained in intracellular vesicles, the Clathrin family of proteins is critical for the organization and activity of the vesicles, and Clathrin plays a key role in intracellular transport by facilitating cargo transport between organelles such as endoplasmic reticulum, golgi apparatus, and endosomes in secretory and endocytic pathways. In view of the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that most clathrin family proteins were up-regulated in sIVs, while sEVs exhibited low expression (fig. 20). This finding suggests that sIVs we have isolated might be involved in communication between different cell compartments within the cell.

4.7 SIVs enrichment analysis of unique proteins

In example 4, we analyzed 106 different proteins from sEVs expressed by three cells sIVs, which are unique proteins expressed by sIVs and can represent sIVs, and we performed gene enrichment analysis on these proteins. In the case of Cellular Component (CC), these proteins are associated with COPII coated endoplasmic reticulum to Golgi transport vesicles, coating vesicles, endoplasmic reticulum to Golgi transport vesicles, endoplasmic reticulum to Golgi middle compartment, transport vesicles membranes, coating vesicles membranes, etc. (fig. 21). Meanwhile, in the Biological Process (BP) category, the terms are glycerophospholipid biosynthesis process, response to endoplasmic reticulum stress, ubiquitin-dependent ERAD pathway, intracellular protein transport and endoplasmic reticulum-to-golgi vesicle-mediated transport, etc. (fig. 22). This result suggests that sIVs isolated is an inherent vesicle component within the cell.

5. Knot (S)

In this example, we used three cells, MSCs, 293T and Hela cells as an example, and applied proteomic techniques to characterize the protein composition of cells sEVs and sIVs. The results indicate that sIVs has a unique protein expression profile, distinct from cells and sEVs; sIVs low expression of exosome markers, high expression of marker proteins of intracellular membrane-rich organelles and Clathrin protein family, which indicate sIVs plays a role in intracellular material transport and mediates intracellular organelle communication; gene enrichment analysis of sIVs directly suggests that sIVs is involved in mass transport between the endoplasmic reticulum and golgi apparatus, which includes forward vesicle-mediated transport of the endoplasmic reticulum to the golgi apparatus, and retrograde vesicle-mediated transport of the endoplasmic body back from the golgi apparatus. These findings strongly suggest that sIVs plays a key role in the intracellular material transport process, particularly in mediating material communication between organelles. Among them, COP-coated vesicles are widely reported to be involved in endoplasmic reticulum initiated intracellular material transport processes. During this process, the correctly folded and assembled proteins in the endoplasmic reticulum are encapsulated into COP-coated transport vesicles, which then detach from the endoplasmic reticulum membrane. Next, the vesicles are de-coated and fused to each other to form a vesicle tubular cluster. The golgi apparatus is responsible for modifying these proteins and lipids received from the endoplasmic reticulum and partitioning them into cell membranes, endosomes and secretory vesicles. Proteins and lipids move in cis to trans direction within the golgi apparatus and this is accomplished by vesicle transport. By proteomic analysis, sIVs was further demonstrated to be involved in intracellular vesicle transport processes, closely related to endoplasmic reticulum, golgi apparatus and COP-coated vesicles.

The above results indicate that sIVs is distinct from sEVs, sIVs plays an important role in intracellular mass transport, sIVs being a unique vesicle population.

Example 6: sIVs has a unique miRNA expression profile

1. Laboratory instrument and materials

1.1 Experimental reagent

PBS, available from Gibco Biolabs (America)

1.2 Laboratory apparatus

Table 6 Experimental apparatus

2. Experimental method

2.1 Data acquisition and analysis

2.1.1 RNA isolation, library preparation and sequencing

RNA degradation and contamination were detected on 1% agarose gels using PBS for resuspension after separation enrichment sEVs and sIVs (prepared in example 2). The RNA purity was checked using a NanoPhotometer cube spectrophotometer. RNA concentrations were measured using the Qubit ™ RNA assay kit in the Qubit cube 2.0 Flurometer. The detection was performed using the RNA Nano 6000 assay kit of the agilent bioanalyzer 2100 system.

2.1.2 Library preparation for Small RNA sequencing

The total RNA amount of 3. Mu.g per sample was taken as an input sample for the small RNA library. Sequencing libraries were generated using the Small RNA Library Prep Set for Illumina cubes of NEBNExt Multiplex software, and index codes were added to assign sequences to each sample. Amplification was performed on a PCR instrument using LongAmp Taq X Master Mix, SR Primer for illumina and index (X) primers. The PCR product was then purified on an 8% polyacrylamide gel (100V, 80 min). The DNA fragment corresponding to 140 to 160bp was recovered and dissolved in 8. Mu.L of elution buffer. Finally, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA HIGH SENSITIVITY CHIPS.

2.1.3 Cluster Generation and sequencing

Index encoded samples were clustered using TruSeq SR Cluster Kit v-cBot-HS (illumina) according to manufacturer's instructions at cBot Cluster Generation System. After cluster generation, sequencing was performed on an Illumina Hiseq 2500/2000 platform, generating a 50bp single-ended reading to prepare the library.

2.1.4 Data analysis

1) Quality control

First, the raw data (raw reads) in fastq format is processed by custom Perl and Python scripts. In this step, clean data is obtained from the raw data by removing the reads containing ploy-N, 5 'terminal contamination, no 3' terminal or insert tag, containing ploy a or T or G or C, and low quality (CLEAN READS). At the same time, the Q20, Q30 and GC content of the raw data were calculated. A range of lengths is then selected from the cleaning readings for all downstream analyses. The use of Bowtie（Langmead B., Trapnell C., Pop M., Salzberg S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genome biology, 2009, 10(3): R25.） to map small RNA tags to reference sequences did not allow for mismatches to analyze their expression and distribution on the reference sequence.

2) Known miRNA alignment

Mapped small RNA tags were used to find known mirnas. With mirbase20.0 as a reference, modified software mirdeep and srna-tools-cli were used to obtain potential mirnas and map secondary structures. The miRNA counts and base bias at the first position of the identified mirnas of a specific length, and the miRNA counts and base bias at each position of all identified mirnas, are obtained separately using custom scripts.

3) Small RNA annotation abstract

All alignments and annotations obtained before were summarized. In previous alignments and annotations, some small RNA tags may be mapped to multiple categories. To ensure that each unique small RNA maps to only one annotation, we follow the following priority rules: known miRNA > rRNA > tRNA > snRNA > snoRNA > YRNA > repeat > gene > novel miRNA.

4) Data analysis

Target gene prediction was predicted using miRanda, miRNA target gene prediction was the intersection of two software, miRanda and RNAhybrid. The differentially expressed miRNA input data is readcount data obtained in miRNA expression level analysis. For samples with biological reproducibility: differential expression analysis was performed for both conditions/groups using the DESeq R package (3.0.3). The P value was adjusted using the Benjamini & Hochberg method. By default, the corrected P value is set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, wien maps, volcanic maps, etc. were plotted on-line using Hiplot and adjusted using Adobe Illustrator.

5) GO and KEGG enrichment analysis

GO enrichment analysis was performed on target gene candidates (hereinafter referred to as "target gene candidates") of differentially expressed mirnas. The GO enrichment analysis adopts GOseq based on the Wallenius non-central hypergeometric distribution, so that the gene length deviation can be adjusted. The statistical enrichment of target gene candidates in the KEGG pathway was tested using KOBAS software.

3. Experimental results

3.1 Relative RNA abundance in sEVs and sIVs of MSCs, 293T and Hela cells

First we analyzed RNA abundance of sEVs and sIVs in MSCs, 293T and Hela cells using a bioanalyzer (fig. 23). The results show that there is no statistical difference in RNA abundance in sEVs and sIVs for the three cells, where the RNA abundance of MSCs is higher than for the other two cells, possibly related to the abundant biological effects of MSCs.

3.2 Small RNA distribution in sEVs and sIVs

Small RNA associated with cells and extracellular vesicles is a hot spot of research in recent years, and particularly microRNA (miRNA) has multiple biological functions and can be used as a biomarker for various diseases. We analyzed the small RNA species in sEVs and sIVs. The comparison and annotation of all kinds of small RNA and total RNA were summarized. Since there is a case where one sRNA is simultaneously compared with several different annotation information, in order to make each unique sRNA have a unique annotation, the small RNA is classified according to the priority order of detection of the known miRNA > rRNA > tRNA > snRNA > YRNA > repeat > gene > novel miRNA, and the proportion of each small RNA to the total RNA is calculated. The results show that YRNA is the predominant RNA in sEVs; in sIVs, miRNA is the predominant RNA. The proportion of miRNA in sEVs of MSCs is 29.15 percent, and the proportion of miRNA in sIVs is 92.52 percent; the proportion of miRNA in sEVs of 293T cells is 13.29%, and the proportion of miRNA in sIVs is 59.42%; the proportion of miRNA in sEVs of Hela cells was 32.22%, and the proportion of miRNA in sIVs was 58.09%. The miRNA content ratio in sIVs of the three cells was significantly more than sEVs (fig. 24). Of the three cells, sIVs of MSCs had more pronounced mirnas, further suggesting that MSCs-derived vesicles, similar to MSCs, may have abundant biological regulatory effects.

3.3 Total miRNA expression profile of sEVs and sIVs

MiRNA has abundant biological regulation and occupies a large proportion in small RNA, so that subsequent analysis of miRNA is carried out, and the result of using Wen diagram to analyze the types of miRNA contained in sEVs and sIVs of three cells shows that 694 miRNA, 803 and 630 miRNA are detected in sEVs of MSCs, 293T cells and Hela cells respectively, and 989 miRNA, 902 miRNA and 795 miRNA are found in sIVs of the three cells (figure 25). There is some overlap in the miRNA species between them, but not exactly the same. MSCs in three cells contained the most abundant mirnas, and sIVs contained more abundant mirnas in svvs and sIVs.

Since sEVs and sIVs contained co-expressed mirnas, the subsequent use of principal component analysis compared the miRNA expression patterns of sEVs and sIVs, which showed a large difference in the miRNA components contained by the two vesicles, the expression patterns were uncorrelated (fig. 26), further demonstrating that sIVs was distinct from sEVs, containing a unique miRNA expression profile.

3.4 High abundance mirnas in sEVs and sIVs

Analysis of the first 10 high abundance mirnas in sEVs and sIVs found that miR-148a-3p, let-7i-5p were at higher expression levels in sEVs of all three cells; let-7f-5p is at a higher expression level in sIVs of the three cells. At the same time, miRNAs such as miR-148-3p, miR-21-5p and miR-100-5p are in higher expression levels in sEVs and sIVs of a plurality of cells (FIG. 27).

3.5 SEVs and sIVs differential expression miRNA assay

To further compare the differences in mirnas between sEVs and sIVs, we performed differential expression analysis on sEVs and sIVs of three cells. The results showed 70 differential mirnas in sEVs and sIVs in MSCs, 22 out of which sIVs was significantly down-regulated compared to sEVs, 48 out of which was significantly up-regulated; there are 167 differential mirnas for sEVs and sIVs in 293T, of which sIVs is significantly down-regulated by 125 and significantly up-regulated by 42 compared to sEVs; there were 120 differential mirnas for sEVs and sIVs in Hela cells, 55 of which sIVs was significantly down-regulated compared to sEVs, 65 of which were significantly up-regulated (fig. 28-29). Further description sIVs differs from sEVs.

3.6 SEVs and sIVs differential expression miRNA target gene enrichment analysis

The way MiRNA plays a biological role is to regulate downstream target genes, so after we compare different miRNAs in each group, the target genes of the miRNAs are respectively subjected to gene enrichment analysis, including GO analysis and KEGG analysis. For convenience of description, we will refer to "target gene differentially expressing miRNA" as "candidate target gene" hereinafter. The results of GO enrichment analysis show that candidate target genes sEVs and sIVs of MSCs are related to intracellular metabolic processes, cells are positioned as organelles related to intracellular membranes, and the like, and molecular functions are related to protein binding, enzymatic metabolic reactions, and the like (fig. 30A); KEGG pathway analysis showed that candidate target genes sEVs and sIVs of MSCs were associated with pathways such as axon guidance, cell differentiation, endocytosis, and immunomodulation (T cell receptor signaling pathway, B cell receptor signaling pathway) (fig. 30B). The results of GO enrichment analysis of 293T cells showed that candidate target genes sEVs and sIVs are also associated with intracellular processes, cell localization to cell membranes, intima system and organelle membranes, etc., and molecular functions are associated with protein binding and enzymatic metabolic reactions (fig. 31A). KEGG pathway analysis showed that candidate target genes sEVs and sIVs of 293T cells were associated with multiple signaling pathways such as CAMP and Ras (fig. 31B). The results of GO enrichment analysis of Hela cells showed that candidate target genes sEVs and sIVs are also associated with intracellular processes, cell localization is also the cell membrane, intima system and organelle membrane, etc., and molecular function is associated with protein binding and enzymatic metabolic reactions (fig. 32A). KEGG pathway analysis showed that candidate target genes were associated with multiple signaling pathways such as synapses and MAPKs (fig. 32B).

4. Knot (S)

In this example, we used MSCs, 293T and Hela cells as targets, and performed detailed analysis of the small RNA composition of sEVs and sIVs using small RNA sequencing technology, and studied the expression pattern of mirnas. Intracellular mirnas are transported to the cytoplasm after being produced in the nucleus, and are involved in the regulation of target genes. Thus intracellular produced mirnas first perform their biological functions by regulating gene expression and are involved in various cellular biological processes such as cell proliferation, differentiation and apoptosis. Many mirnas are found to be specifically expressed in different types of stem cells, regulating the process of cell differentiation and maturation of specific cell lines. Other mirnas may promote or inhibit cell death signaling pathways, affecting cell survival and apoptosis. They can maintain cell homeostasis by modulating apoptosis-related genes such as BCL2 family members, caspases and p53, etc. In cells, mirnas can act as signal pathway modulators to modulate cellular biological processes, such as growth factor signaling, coping with oxidative stress and inflammatory responses, and the like. They can target key molecules in a particular signaling pathway, affecting the entire signaling pathway. The experimental result shows that sIVs is rich in rich miRNA, so that sIVs is presumed to play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis and signal paths, and has great application potential. Research results show that sIVs shows a unique miRNA expression profile, which is significantly different from sEVs, and the miRNA content in sIVs is more abundant. This finding suggests sIVs has potential biological regulatory effects and may promote the communication of information between different organelles within a cell. By performing candidate gene enrichment analysis on mirnas differentially expressed in sEVs and sIVs, we further found that sIVs was in close association with endomembrane-like organelles. Taken together, sEVs and sIVs differ in small RNA composition, especially miRNA. Enrichment analysis for mirnas further demonstrated that sIVs plays an important role in intracellular mass transport. Compared with sEVs, sIVs contain more diverse miRNAs and are likely to have more abundant biological functions. These findings provide important clues for a thorough understanding of sIVs's function in cell biology.

Example 7: sIVs has unique lipidomic characteristics

1. Laboratory instrument and materials

1.1 Experimental reagent

Table 7 Experimental reagent

1.2 Laboratory apparatus

Table 8 laboratory apparatus

2. Experimental method

2.1 Metabolite extraction

The enrichment sEVs and sIVs (prepared in example 2) were separated and resuspended using PBS. 200. Mu.L of water was added to the vessel followed by 480. Mu.L of an extract made up of MTBE and MeOH mixed in a 5:1 ratio and containing internal standard material. The mixture was rapidly placed in a liquid nitrogen tank and frozen for 1 minute, then taken out for thawing, and mixed by a vortex mixer for 30 seconds to homogenize the solution. 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℃for 1 hour. Then, the sample was centrifuged at 3000rpm (centrifugal force 900 Xg, radius 8.6 cm) at 4℃for 15 minutes to separate the sample. 300. Mu.L was removed from the supernatant, transferred to an EP tube, and dried under vacuum. To the dried sample was added 100 μl of the complex solution (DCM: meoh=1:1), vortexed for 30 seconds, and sonicated again in an ice-water bath for 10 minutes. Finally, the mixture was centrifuged at 13000rpm (centrifugal force 16200 Xg, radius 8.6 cm) at 4℃for 15 minutes, and 75. Mu.L of the supernatant was transferred to a sample bottle and prepared for on-machine detection.

2.2 Metabolite detection

The target compound was chromatographed using a Vanquish ultra performance liquid chromatograph using Waters ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm, 1.8 μm) liquid chromatography column. For liquid chromatography, phase A is a 40% aqueous solution containing 10mmol/L ammonium formate and a 60% acetonitrile solution; phase B was a solution of 50mL/1000mL (10 mmol/L) of aqueous ammonium formate in 10% acetonitrile and 90% isopropanol. We used the following gradient elution procedure: 0-1.0 min, 40% B;1.0 to 12.0min, linearly increasing to 100% B; 12.0-13.5 min, maintaining 100% B; 13.5-13.7 min, linearly reducing to 40% B;13.7 to 18.0min, and 40% B is maintained. The flow rate of the mobile phase was set to 0.3mL/min, the column temperature was 55deg.C, the sample pan temperature was 4deg.C, and the sample injection volume was 2 μL (positive and negative ion modes).

Meanwhile, primary and secondary mass spectrometry data acquisition was performed using Thermo Q Exactive HFX mass spectrometers under the control of Xcalibur control software (version: 4.0.27, thermo). The specific parameters are as follows: SHEATH GAS flow rate was set to 10Arb,Capillary temperature at 350 ℃, full MS resolution at 120000, MS/MS resolution at 7500,Collision energy at 10/30/60 in NCE mode, and spray Voltage at 4kV (positive ion mode) or-3.8 kV (negative ion mode).

2.3 Data analysis

The mass spectra were originally converted to mzXML format using ProteoWizard software. The retention time correction, peak identification, peak extraction, peak integration, peak alignment were again performed using XCMS, minfrac set to 0.5, and cutoff set to 0.3. Lipid identification was performed using XCMS software, self-written R package and lipidblast database. The letter drawings are drawn online using Hiplot and adjusted using Adobe Illustrator.

3. Experimental results

The ionization source of the Orbitrap platform is electrospray ionization, and has two ionization modes, namely a positive ion mode (positive ion mode, POS) and a negative ion mode (negative ion mode, NEG), when a metabolome is detected, the two modes are combined, so that the metabolite coverage rate is higher, the detection effect is better, and when data analysis is carried out, one ion mode is generally selected, and the study is carried out by taking the positive ion mode as an example.

3.1 Ratio of each metabolite in sEVs and sIVs of MSCs, 293T and Hela cells

The secondary spectrogram of lipidomics is occasional, so that lipid substances identified in all groups in a set of comparison information are only plausible. Therefore, we performed classification statistics on the metabolites identified by different cells according to the chemical classification attribution information, and the contents of various metabolites are shown in fig. 33. MSCs identified 31, 293T cells identified 43, and Hela cells identified 44. There were differences in the expression levels of various lipid species in sEVs and sIVs. PC and PE are common lipid components of cell membranes, and the results in fig. 33 show that the ratio of PE to PC is high in vesicles of three cells, indicating the presence of large numbers of biofilm structures at sEVs and sIVs.

3.2 Characterization of Total lipid expression in sEVs and sIVs of MSCs, 293T and Hela cells

The metabolomic data has the characteristic of high flux, and the main component analysis can effectively highlight the overall distribution trend of the metabolomic data and the difference degree of samples among groups. The results showed that MSCs, 293T and Hela cells sEVs and sIVs have different lipid profiles (fig. 34), i.e. sIVs is a unique population of vesicles, with significantly different lipid expression patterns, as distinguished from sEVs.

3.3 Differential lipid expression signatures in sEVs and sIVs of MSCs, 293T and Hela cells

The heat map can intuitively show the overall distribution of the differences of metabolites among groups, and the result of screening the different metabolites is visualized in the form of the heat map. The results of sIVs set versus sEVs set are shown in figure 35. sIVs of non-cancer cells highly express conventional biomembrane structural lipids such as PC, PI and PE, but the lipid expression pattern of sIVs of cancer cells Hela cells is different from that of non-cancer cells, and can be related to the abnormal lipid metabolism of cancer cells.

3.4 Differential lipid content variation degree and classification information in sEVs and sIVs of MSCs, 293T and Hela cells

The lipid profile is visualized using the degree of change in the content of the metabolites and the classification information, and the results of sIVs versus sEVs are shown in fig. 36, where each column represents a metabolite. PC, PI, PE, PG and OxPI were significantly higher expressed in MSCs in sIVs, with PC being more than 200-fold higher expressed in sIVs; PE, cer/AS, cer/AP, cer/ADS, PI, PC and the like are remarkably high-expressed in sIVs in 293T cells, wherein PE is high-expressed by more than 900 times in sIVs; SHexCer, CE, PI and HexCer/NS et al were significantly high expressed in Hela cells in sIVs, with SHexCer being 400-fold more highly expressed in sIVs.

4. Knot (S)

Lipidomics identify and quantify various lipid molecules, lipids are classified into eight broad categories, including fatty acyl, glycerolipid, phospholipid, sterol lipid, propenol lipid, sphingolipid, glycolipid, and polyketone. Cell membranes mainly contain various phospholipids, which can be further divided into glycerophospholipids and sphingomyelins, which have significant differences. Glycerophospholipids are mainly located in the inner leaflet of phospholipid bilayer in cell membrane, and together with cholesterol constitute the main component of cell membrane, and sIVs in this example contains more glycerophospholipids such as PC and PE. Sphingomyelin is a type of phospholipid containing sphingosine groups, which is located on the outer leaflet of cell membranes and is mainly involved in neuronal activity and signaling, and sEVs in this example contains more sphingomyelin, such as SM. In addition, glycerophospholipids are involved in many other physiological processes in the body, such as energy metabolism, hormone synthesis, etc.; while sphingomyelin plays a relatively minor role in these processes. Overall, MSCs, 293T and Hela cells sEVs and sIVs have different lipid profiles. sIVs the expression levels of PC and PE were higher, wherein PC was expressed 200 times higher in sIVs. Wherein PC is also called lecithin, is known as a third nutrient which is juxtaposed with protein and vitamin, and has a plurality of important roles in biology. Lecithin can increase neurite growth of neurons, promote brain development, enhance memory, and prevent senile dementia. In addition, PE is one of the main molecules constituting the skeleton of a biological membrane. Its unique structure, including a phosphate group, a glycerol, an acyl group and an ethanolamine, enables it to form stable non-lamellar as well as multilamellar liposome vesicles in biological membranes. This structure provides a stable foundation for the biofilm, helping to maintain the normal structure and function of the cell.

Interestingly, both PC and PE with high expression sIVs are glycerophospholipids, the endoplasmic reticulum is the site of glycerophospholipid synthesis, so sIVs containing more glycerophospholipids is reasonable, further confirming that sIVs is an intracellular component, mediating the transport of substances within the cell and communication between organelles. sEVs, however, contains more sphingomyelin, sEVs is derived from the invagination of the cell membrane and is secreted out of the cell via the cell membrane, and thus contains more of the outer cell membrane components. This also indicates that sIVs lacks an external membrane structure, and that the distinction of various lipids distinguishes sIVs from sEVs. Previous studies have shown that intracellular transport of proteins and lipids is related to membrane bending and lipid profile effects. Glycerophospholipids can regulate the curvature and fluidity of membranes by regulating chain length and acting synergistically with cholesterol, conferring sIVs more viability, thus continuing to participate in intracellular membrane fusion and fission events.

In this example, the lipidomic data further verifies sIVs is distinct from sEVs, providing an important basis for the deep exploration of the unique properties of sIVs in cell and tissue compatibility.

Example 8: cells cultured in vitro and retinal tissue in vivo have higher absorption efficiency to sIVs

1. Laboratory instrument and materials

1.1 Experimental reagent

Table 9 Experimental reagent

1.2 Laboratory apparatus

Table 10 laboratory apparatus

2. Experimental method

2.1 Cell culture

Human HRMECs was purchased from Angioproteomie, usa. Preparation of complete cell culture medium: to 93ml of ECM minimal medium, 5ml of fetal bovine serum, 1ml of growth factor and 1ml of penicillin-streptomycin were added to give a complete medium containing 5% serum. RPE cell complete medium preparation: to 44.5ml of DMEM minimal medium, 5ml of fetal bovine serum and 0.5ml of penicillin-streptomycin were added to obtain a complete medium containing 10% serum. The cell growth density is checked by using a low-magnification microscope, and round, elliptic or polygonal flat cells are observed by using a high-magnification microscope, and the cytoplasm contains rich components and has small vacuoles in the cells. Cells were inoculated in culture flasks and cultured in a 5% CO ₂ incubator at 37 ℃. The medium was changed every 3 days. When cell fusion reached 80%, passaging was performed at a 1:2 ratio of subcultures, and experiments were performed using cells of P3 to P5.

2.2 Laboratory animals

Healthy male C57BL/6 mice, 8 weeks old, body weight 21-22g, grade SPF, purchased from Beijing vetton Lihua corporation (animal production license: SCXK (Beijing) 2016-0006). All experimental animals were normally fed at room temperature with light and dark times of 12 h, respectively. The animal raising environment and the experimental operation content meet the regulations of the national institutes of sciences and technology (SOG) and the national institutes of animal ethics committee license (ethics number: TJYY 2019091124) is obtained. Mice were grouped according to the random number table method for subconjunctival and intravitreal injections.

2.3 DiD markers sIVs and sEVs were co-cultured with cells

The vesicles were incubated with lipid-soluble tracer DiD solution for 30 min at 37 ℃. Excess DiD was removed by Amicon cube Ultra centrifuge filters. Cells were seeded into pre-placed circular plates on 24-well plates and incubated for 24 hours. DiD-labeled vesicles were added to the cell culture medium and incubated at 37℃for various times: 3 hours, 12 hours, 24 hours or 48 hours. After removal of the medium, the cells were washed with PBS and at room temperature. Cells were fixed with 4% PFA for 10min and then incubated with 0.1% Triton X-100 for 5 min at room temperature to allow cell infiltration. Subsequently, cells were stained with CoraLite% Plus 488 conjugated Phalloidin antibody for 30 min at room temperature. Nuclei were labeled with DAPI. Finally, cells were imaged using a confocal laser scanning microscope and the amount of endocytic vesicles was analyzed by Image J.

2.4 Subconjunctival and intravitreal injections of DiD labeled sIVs and sEVs

As before, vesicles were stained with DiD. DiD-labeled vesicles were injected under the conjunctiva of mice in a volume of 5. Mu.L and a mass of 3. Mu.g. 24 hours and 48 hours after subconjunctival injection, the eyeballs were collected for observation. DiD-labeled vesicles were infused into the vitreous cavity of mice in a volume of 1. Mu.L and a mass of 1. Mu.g. Eyeballs were collected for evaluation 8 hours and 48 hours after the vitreous injection.

2.5 Retinal sections

Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. Retinal sections were fixed with PFA at room temperature for 15min, followed by washing the slides with PBS and staining of nuclei with DAPI. Finally, the tablets were capped with an anti-fluorescent decay cap and observed using a confocal laser scanning microscope.

2.6 Ultrasonic drug delivery

Rapa-sEVs and Rapa-sIVs are prepared by ultrasonic methods. Rapa with sEVs or sIVs (prepared in example 2) were combined in a 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 30 second pulse and 30 second pause). Subsequently, the mixture was incubated at 37 ℃ for 1 hour to promote membrane recovery. After the sonication process was completed, the free Rapa was removed by ultrafiltration centrifugation.

2.7 High Performance liquid chromatography detection sIVs and Rapa in sEVs

The Rapa concentration in Rapa-sEVs or Rapa-sIVs was assessed using high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). A standard curve was generated from a range of Rapa concentrations (ranging from 0 to 50. Mu.g/mL). 20. Mu.L of standard and sample solutions were injected into the HPLC system for analysis. Standard curves were plotted using the average peak area as the abscissa and Rapa concentration as the ordinate, and regression equations were calculated. Acetonitrile was added to Rapa-sEVs or Rapa-sIVs to precipitate the protein of sEVs or sIVs and extract Rapa. After centrifugation, the supernatant was analyzed for Rapa. The chromatographic column used was a C18 reverse phase column. The mobile phase consisted of HPLC grade acetonitrile and water (Vacetonitrile/Vwater =65/35), flow rate was 1ml/min. The detection wavelength was 278nm and the column temperature was maintained at 62 ℃. According to the formula: (in-package drug)/(in-package drug + sEVs or sIVs total mass) ×100% and the load capacity (%) was calculated. Encapsulation efficiency (%) was calculated using the following formula: (amount in drug encapsulation)/(total drug amount) ×100%.

2.8 LC-MS detection of Rapa in retina

The Rapa standard was measured precisely to a concentration of 5mM, dissolved in methanol, and prepared as a Rapa standard intermediate stock solution having a concentration of 50. Mu.M. About 2mg of the internal standard carbamazepine standard is taken and dissolved in methanol to prepare a carbamazepine standard stock solution with the concentration of 2.00 mg/ml. Accurately transferring a proper amount of Rapa standard substance intermediate stock solution, and serially diluting with methanol to obtain Rapa standard line working solutions with concentrations of 2000, 1000, 500, 200, 50.0, 20.0, 10.0 and 5.0nM in sequence, wherein the working solutions are used for configuring standard curve samples in a dosing analysis batch; taking 50 mu l of methanol in a 1.5ml EP pipe, adding 5 mu l of standard curve working solution into the pipe, adding 10 mu l of internal standard working solution 2, fully vortex mixing, adding 50 mu l of methanol again, and performing sample injection analysis after vortex mixing. The Rapa standard curve concentrations in the dosing sample analysis batch were: 200. 100, 50, 20, 5.0, 2.0, 1.0, 0.5nM. Taking out frozen mouse retina samples, adding 100uL of chromatographic pure methanol into each tube, adding grinding beads, carrying out homogenization treatment in a tissue grinder, grinding for 2min at 50Hz, stopping for 5s every 30s, taking 50uL of homogenate in a 1.5ml EP tube, adding 5 mu l of methanol solution into the tube, 10 mu l of internal standard working solution 2, fully and uniformly mixing by vortex, adding 50 mu l of methanol, vortex for 1min of extractant, 12000rpm, centrifuging for 10min, and taking 100 mu l of supernatant for sample injection analysis. Mass spectrometry conditions: air curtain gas: 25 psi, collision gas (CAD): medium, ion source voltage (spray voltage): 5000 V, ion source temperature: 550 ℃, atomizing gas (GS 1): 60 psi, auxiliary heating gas (GS 2): 55psi.

TABLE 11 liquid phase conditions

3. Statistical treatment

4. Experimental results

4.1 Endocytosis sIVs capacity better than sEVs

To assess the phagocytic capacity of cells against both types of vesicles, RPE cells and HRMECs were selected for co-culture with MSC-sEVs (abbreviated as sEVs in this example) and MSC-sIVs (abbreviated as sIVs in this example). Briefly, equal amounts of the di-labeled vesicles were incubated with cells, and at various time points, the distribution of the di was observed using confocal microscopy to observe the uptake of vesicles by the cells. The results showed that after 3 hours of co-culture, cells began internalizing sEVs and sIVs and their uptake continued to increase over time (fig. 37A, 37B, 38A, 38B). At 24 hours, the amount of sEVs and sIVs internalized by the cells peaked, followed by a gradual decrease over 24-48 hours (fig. 37C, fig. 38C). Notably, in RPE cells and HRMECs, the internalization rate of cell pair sIVs consistently exceeded sEVs over a period of 12 to 48 hours (fig. 37C, fig. 38C). This indicates that cells cultured in vitro have high uptake efficiency for sIVs.

4.2 Retinal endocytosis sIVs Capacity over sEVs

To assess the phagocytic capacity of the retina for both types of vesicles, the eye was administered an equal amount of di-labeled vesicles by two methods: subconjunctival and intravitreal injections. Eyeball samples were collected at different time points, then frozen sections were prepared, and vesicle uptake in the retina was observed using confocal microscopy. The results showed that, 24 hours after subconjunctival injection, sIVs penetrated the sclera and entered the subretinal space, while sEVs accumulated between the conjunctiva and sclera, less sEVs reached the subretinal area (fig. 39A). After 48 hours, the retina showed significant uptake of sEVs and sIVs (fig. 39C). Specifically, sEVs are distributed in the RPE layer, while sIVs reaches deeper locations, widely distributed in the outer nuclear, inner nuclear and ganglion cell layers. After intravitreal injection of the di-labeled vesicles, the vesicles diffuse from the vitreous cavity to the retina. The results showed that, 8 hours after injection, sEVs was distributed within the vitreous cavity, not yet reached the retina, while sIVs had reached the entire retinal layer and was internalized by the ganglion cell layer and the inner core layer cells, with higher sIVs accumulation observed on the RPE layer (fig. 39B). 24 hours after intravitreal injection, the retina effectively absorbed sEVs and sIVs, both types of vesicles being diffusely distributed throughout the retina (fig. 39B). sIVs exhibited more significant uptake compared to sEVs (fig. 39D) and was widely distributed across all retinal layers, including ganglion cell layers, inner plexiform layers, inner nuclear layers, outer plexiform layers, outer nuclear layers, and RPE layers (fig. 39B).

4.3 SIVs has better drug carrying capacity than sEVs

To further investigate the ability of the retina to absorb the vesicle contents, we use ultrasound to encapsulate lipophilic small molecules Rapa into vesicles. Transmission electron microscopy images showed that sEVs and sIVs maintained their structural integrity after both sonication and drug entrapment (fig. 40A), without any significant change in vesicle size (fig. 40B). The amount of Rapa carried by both types of vesicles after sonication was assessed by HPLC and the characteristic peaks of Rapa are shown in fig. 41A. Calculations have shown that sIVs exhibits a considerably higher packaging and loading efficiency than sEVs (fig. 41B and 41C).

4.4 Retina absorbs sIVs the drug-loaded ability is stronger

Based on the packaging and load efficiency, the Rapa amount carried by sEVs and sIVs was calculated. By supplementing PBS, two vesicles carrying equal amounts Rapa and the same amount of Rapa pure were obtained. The three solutions were then adjusted to equal volumes and injected into the subconjunctival space and vitreous cavity of the mice, respectively. At various time points, retinal samples were collected to assess Rapa content. The results of the study showed that 24 hours after subconjunctival injection of both drug-loaded vesicles, the amount of Rapa absorbed from the vesicles by the retina was higher than that of Rapa pure product, compared to direct injection Rapa. For 48 hours, the retina absorbed significantly more Rapa from sIVs than from sEVs, and this pattern continued for the fifth day (fig. 42A). Meanwhile, 24 hours after intravitreal injection of both drug-loaded vesicles, analysis of the collected retinal samples showed that sIVs delivered more Rapa amounts to the retina than sEVs and pure Rapa (fig. 42B).

The results show that sIVs has larger drug carrying capacity, can carry more drugs, and the drugs carried by sIVs can be absorbed and internalized by tissues more. sIVs are good drug carriers.

5. Knot (S)

This example shows that endocytosis of sIVs by cells cultured in vitro is better than sEVs. The finding that the endocytic capacity of the retina to sIVs was observed to be better than sEVs by subconjunctival and intravitreal injection of mice fully demonstrates the good histocompatibility of sIVs. Subsequently, we loaded the liposoluble drug Rapa into sIVs and sEVs using ultrasound drug delivery techniques, which indicated that sIVs had higher drug delivery and that the vesicle wrap Rapa improved the absorption of Rapa by the retina. The retina has higher absorptivity to Rapa mounted on sIVs. This indicates that sIVs is easily absorbed by tissues and has excellent capacity to load lipid-soluble small molecule drugs. The results not only prove the feasibility of sIVs as a medicine carrier, but also lay a solid foundation for the application of sIVs in the treatment of posterior segment diseases after medicine loading.

Example 9: sIVs application of protein medicine as medicine carrier

In this example, sIVs was further tested as a carrier-loaded drug using interleukin 10 (IL-10) as an example of the drug.

1. Experimental method

1.1 Ultrasonic drug-loaded Interleukin 10 (IL-10)

MSC-sIVs (prepared in example 2, abbreviated as sIVs in this example) is taken, IL-10 drug solution is added in sequence according to mass ratio gradient (1:20, 1:10, 1:5, 1:1), and ultrasonic treatment is carried out after mixing evenly: 30s on/30 s off,6 cycles. After the end of the sonication, the liquid was centrifuged through a 100kDa ultrafiltration tube to remove free drug.

1.2 Detection of efficiency of sIVs to carry IL-10 Using enzyme-Linked immunosorbent assay (ELISA)

The IL-10-loaded sIVs was added to ELISA kit well plates, and the corresponding standard control was added, and incubated at room temperature for 1: 1 h. The corresponding volume of primary antibody was added and the plate was washed three times after incubation at room temperature of 1 h. The corresponding volume of secondary antibody was added and the plate was washed three times after incubation at room temperature of 1 h. Adding a corresponding volume of substrate for color development, and after incubation for 20min at room temperature, adding a corresponding volume of stop solution for color development. The microplate reader 562 is excited to detect absorbance values. Finally, the expression level of IL-10 in different samples was calculated.

1.3 T cell proliferation in vitro experiment (CFSE method)

96-Well plate pretreatment (plating): diluting the CD3 antibody in PBS (phosphate buffer solution), wherein the concentration is 10 mu g/ml, 100 mu l of PBS is added to each hole of a 96-well plate, 100 mu l of PBS is added to the outer ring hole of the 96-well plate to prevent excessive evaporation, the solution is incubated at 4 ℃ overnight or in a cell incubator at 37 ℃ for 2 hours, the antibody is sucked out, and the PBS is washed twice for later use.

Total CD4 ⁺ T cells are adjusted to 5ml, placed in a centrifuge tube, 1 μl CFSE storage solution (5 mmol/L) is added, the mixture is quickly mixed, incubated at room temperature and in a dark place for 10min, RPMI1640 medium containing 10% FBS in the same volume is added to stop staining, after centrifugation, the complete medium (configuration: 90%1640 basal medium+10% FBS+2 μg/ml CD28 antibody+100U/ml double antibody+50 [ mu ] M beta-mercaptoethanol) is resuspended, 3x10 ⁵/well cells are plated in a pretreated 96-well plate, a blank control group and a sIVs treatment group loaded with IL-10 are arranged, and three auxiliary wells are arranged in each group. After 72h incubation, the harvested cells were stained with CD4-APC/CD40L-PerCp antibody, and the flow-through was examined for CFSE fluorescent expression and CD40L ⁺ T cell ratio changes.

2. Experimental results

2.1 Drug loading rate of sIVs loaded with IL-10

The IL-10 pure product package is loaded into sIVs by an ultrasonic method, and after ultrafiltration washing, the ELISA detection is carried out on sIVs after collecting the IL-10 package, and the result shows that the drug loading rate of three independent experiments is higher than 50 percent (figure 43).

2.3 IL-10 loaded sIVs has significant inhibitory effect on T cell proliferation in vitro

SIVs after IL-10 inclusion was added to T cells cultured in vitro, and proliferation of the T cells was examined by flow cytometry. The results showed that sIVs, after IL-10 entrapment, significantly inhibited T cell proliferation in vitro compared to the control group (fig. 44 and 45).

3. Knot (S)

The experimental result of the combination example 7 shows that sIVs prepared by the invention has good capacity of loading fat-soluble micromolecular medicaments and water-soluble proteins as a medicament carrier, and can greatly improve the delivery efficiency.

Examples 1-3 above demonstrate the sIVs collection procedure and parameter optimization procedure. Example 4 demonstrates sIVs physical properties from the point of view of electron microscopy morphology, particle size, characteristic protein and live intracellular distribution, and demonstrates that sIVs has high thermal stability. Examples 5-7 demonstrate the uniqueness of sIVs and the significant differences between sIVs and sEVs from the protein, nucleic acid and lipid point of view, respectively. Example 8 shows that cells cultured in vitro and retinal tissue in vivo have a higher uptake efficiency for sIVs. Examples 8 and 9 demonstrate sIVs that drugs such as small molecule drugs, protein drugs can be loaded and delivered to cells or tissues with significantly better drug loading, encapsulation and delivery than exosome-based sEVs. The above nine examples show that the method for collecting sIVs in vitro according to the present invention is reliable, and sIVs collected according to the present invention is a unique group of intracellular vesicles, has a composition different from the biological macromolecules of sEVs reported in the prior art, such as proteins, nucleic acids and lipids, and has excellent cell tissue affinity and capability of carrying and delivering drugs, and has very good application and research values in the medical field.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. A method for preparing a nanovesicle of intracellular origin, comprising the steps of:

The amplitude of the ultrasonic treatment in the step (1) is 20% -25%, and the time of the ultrasonic treatment is 10-20s;

the cells in the step (1) are isolated cells obtained after culturing, digesting and washing;

the number of times of the centrifugal treatment in the step (2) is two, and the respective parameters are as follows:

1000-3000g,5-20 minutes;

10000-30000g,20-40 minutes;

the parameters of the ultracentrifugation treatment in step (3) include 100000-180000g,50-100 minutes;

the cells are derived from a mammal.

2. The method of claim 1, wherein the amplitude of the ultrasonic treatment in step (1) is 20%; and/or the ultrasonic treatment is carried out for 15s, on 2s and off 2s.

3. The method of claim 1, wherein the centrifugation is performed twice in step (2), and the respective parameters are:

2000g,10 min;

20000g,30 min.

4. The method of claim 1, wherein the parameters of the ultracentrifugation treatment in step (3) include 150 g,70 minutes.

5. The method of claim 1, wherein one or more of the ultrasonic treatment, the centrifugal treatment, and the ultracentrifugation treatment is performed at 0-5 ℃.

6. The method of any one of claims 1-5, wherein the cell is at least one of a stem cell, a cardiomyocyte, an epithelial cell, a macrophage, a lymphocyte, a tumor cell, a fibroblast, a glial cell, and a dendritic cell.

7. Vesicles prepared according to the method of any one of claims 1 to 6 having an average particle size of 50 to 100nm.

8. Use of vesicles prepared according to any one of claims 1 to 6 as a pharmaceutical carrier in the manufacture of a medicament for the prophylaxis and/or treatment of a disease.

9. The use according to claim 8, wherein the disease is an ocular disease.

10. A method of preparing a drug-loaded vesicle, comprising loading a vesicle prepared according to any one of claims 1-6 as a drug carrier.

11. The method of claim 10, wherein the method of preparation of the drug-loaded vesicles is selected from the group consisting of: one or more combinations of genetic engineering, chemical synthesis, viral vector, sonication, electroporation, and co-incubation.

12. A drug-loaded vesicle, characterized in that it uses the vesicle produced by the method of any one of claims 1 to 6 as a drug carrier.

13. The drug-loaded vesicle of claim 12, wherein said drug is selected from the group consisting of: rapamycin, doxorubicin, paclitaxel, carmustine, curcumin, interleukin 10, bevacizumab, ranibizumab, aflibercept, busizumab, and fariximab.