CN113952362A

CN113952362A - Use of induced extracellular vesicles for the preparation of a preparation for prolonging the lifespan of a mammal or for treating or preventing aging

Info

Publication number: CN113952362A
Application number: CN202010641458.0A
Authority: CN
Inventors: 寇晓星; 施松涛
Original assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Current assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Priority date: 2020-07-06
Filing date: 2020-07-06
Publication date: 2022-01-21

Abstract

The application discloses the use of induced extracellular vesicles for the preparation of a formulation for prolonging the lifespan of a mammal or for treating or preventing aging. The present application demonstrates, through several examples, that induced extracellular vesicles have anti-aging effects, and can prolong the life of mammals, and have the effect of reducing alopecia senilis. Furthermore, the application provides the use of induced extracellular vesicles for the preparation of an anti-ageing, and/or repairing, and/or regenerating formulation of the skin and/or skin appendages.

Description

Use of induced extracellular vesicles for the preparation of a preparation for prolonging the lifespan of a mammal or for treating or preventing aging

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of induced extracellular vesicles in preparation of a preparation for prolonging the life of mammals or treating or preventing aging.

Background

With the coming of the global aging society, the prevention and treatment of the health and senile diseases of the elderly have become important public health problems facing the international society. China is the country with the most population in the world and is also the country with the most population for the elderly. The 'aging society' is a serious problem facing China. To solve the troubles brought by aging society, anti-aging medicine is beginning to rise. Exploring aging mechanisms. The search for new targets and new therapies for anti-aging becomes a major topic of life science.

Aging is a life-long deterioration of the physiological totality caused by external stimuli and internal processes. The aging of the body is the final result of cell aging, and the aging is also the manifestation of insufficient numbers of stem cells and body cells. In order to delay aging and maintain homeostasis in the body, continuous tissue renewal and regeneration are required.

Extracellular Vesicles (EVs) are nanoscale carriers containing proteins, nucleic acids, and various cytokines secreted by cells. Extracellular vesicles can act on target cells through endocrine or paracrine modes, and play an important role in the processes of intercellular substance transfer and information exchange. Researches show that the information exchange mediated by the extracellular vesicles plays an important role in regulation and control in the physiological or pathological process of an organism, and relates to immunoregulation, tumor growth, angiogenesis, injury repair and the like. Research in this area is currently focused mainly on the exosomes (exosomes) direction. Exosomes are extracellular vesicles with a diameter of about 30-150nm, and contain RNA, lipids, proteins, and other components. Exosomes are widely involved in various physiological/pathological regulation of the body and can be used as diagnostics, therapeutics and prognostic assessments of a variety of diseases. To date, Mesenchymal Stem Cells (MSCs) are considered to be the most potent cells for producing exosomes. A plurality of researches find that the exosome from the MSCs can simulate the biological function of the MSCs and plays an important regulation and control role in promoting the growth and differentiation of cells, repairing tissue defects and the like. Therefore, in recent years, cell vesicle therapy based on exosomes derived from MSCs has been remarkably developed. However, the current cell vesicle therapy based on exosome still has many problems, which are mainly shown in that the extraction and purification process of exosome is complex, time-consuming, high in requirement on equipment and reagents, low in physiological exosome yield and the like, and the clinical transformation and application of exosome therapy are limited by the defects.

There is no research on the use of effective extracellular vesicles for the preparation of a medicament for prolonging the lifespan of a mammal or treating or preventing aging-related diseases.

Disclosure of Invention

In one aspect, the application provides the use of induced extracellular vesicles for the manufacture of a formulation for prolonging the lifespan of a mammal or for treating or preventing aging.

In some embodiments, the induced extracellular vesicles achieve an extended lifespan or treatment or prevention of aging in a mammal by restoring proliferation and/or differentiation of damaged cells.

In some embodiments, the formulation is a formulation for treating or preventing obesity in the elderly.

In some embodiments, the formulation is a formulation for treating or preventing alopecia due to aging.

In some embodiments, the formulation is a formulation for treating or preventing a spleen enlargement.

In some embodiments, the formulation is a formulation for treating or preventing osteoporosis, bone loss, or bone aging.

In some embodiments, the formulation is selected from a pharmaceutical formulation or a nutraceutical formulation.

In some embodiments, the induced extracellular vesicles are used to reduce the weight of an elderly individual.

In some embodiments, the induced extracellular vesicles are used to reduce hair loss. In one embodiment of the present application, the induced extracellular vesicles derived from the mesenchymal stem cells can significantly improve the hair loss of 24-month-old mice, and have a more significant effect than the mesenchymal stem cells themselves.

In some embodiments, the induced extracellular vesicles are used to reduce spleen weight or volume.

In some embodiments, the induced extracellular vesicles are used to increase bone density.

In some embodiments, the induced extracellular vesicles are used to increase bone volume fraction.

In one aspect, the application provides the use of induced extracellular vesicles for the preparation of a formulation for the treatment or prevention of alopecia senilis.

In one aspect, the present application provides the use of induced extracellular vesicles for the preparation of an anti-aging, and/or repair, and/or regeneration formulation for skin and/or skin appendages.

In some embodiments, the skin is the epidermis, dermis, or subcutaneous tissue.

In some embodiments, the skin appendage is hair, sweat glands, sebaceous glands, nails, or toenails.

In some embodiments, the formulation is a formulation for treating or preventing hair loss, or a formulation for promoting hair regrowth.

In some embodiments, the formulation is one that promotes repair and/or regeneration of a skin wound or scar.

In some embodiments, the induced extracellular vesicles are produced from stem cells selected from mesenchymal stem cells or induced pluripotent stem cells.

In some embodiments, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells, skin mesenchymal stem cells.

In some embodiments, the stem cell is selected from one or more of induced pluripotent stem cells, blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, oral mesenchymal stem cells, skin mesenchymal stem cells.

In some embodiments, the stem cells are selected from one or more of induced pluripotent stem cells, blood stem cells, or bone marrow mesenchymal stem cells.

In some embodiments, the induced extracellular vesicles are induced by the addition of staurosporine, uv irradiation, starvation, or heat stress or a combination thereof to induce apoptotic production of mesenchymal stem cells.

In some embodiments, the induced extracellular vesicles are produced by inducing apoptosis of mesenchymal stem cells by addition of staurosporine.

In some embodiments, the concentration of staurosporine is greater than or equal to 1 nM; preferably, it is 1-15000 nM; preferably 200-; preferably, 250-1000 nM; preferably 500-1000 nM. In addition, the concentration of staurosporine can also be 280-9000 nM; may also be 230-; and can also be 500-1000 nM; and can also be 500-900 nM; and may be 500 and 800 nM.

In some embodiments, the diameter of the inducing extracellular vesicles is 0.45 μm or less. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.45 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.1-0.45 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.1-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.3 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.2 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.4 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.38 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.35 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.32 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.3 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.25 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.05-0.22 μm. In some embodiments, the diameter of the inducing extracellular vesicles is 0.15-0.22 μm. In some embodiments, the diameter of the inducing extracellular vesicles may also be 0.15-0.45 μm, and may also be 0.2-0.3 μm.

In some embodiments, the induced extracellular vesicles have the marker Syntaxin 4. In some embodiments, the inducible extracellular vesicle high expression marker Syntaxin 4. In some embodiments, the marker Syntaxin 4 of the induced extracellular vesicles is expressed higher than MSC or exosomes. In some embodiments, the marker Syntaxin 4 is expressed in an amount 3-6 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker Syntaxin 4 is expressed in an amount 3.5-5 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker Syntaxin 4 is expressed in an amount 4.45 times the amount of expression of Syntaxin 4 in exosomes derived from mesenchymal stem cells. In some embodiments, the marker further comprises one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the marker is a combination of Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5. In some embodiments, the inducible extracellular vesicle overexpression markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 of the induced extracellular vesicles are expressed in higher amounts than the MSCs or exosomes. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in the induced extracellular vesicles in an amount of 1-2 fold, 2-3 fold, 1-3 fold, and 3-4 fold, respectively, relative to the amount of expression of the markers in exosomes derived from mesenchymal stem cells. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in the induced extracellular vesicles in an amount of 1.5-2 fold, 2.5-3 fold, 1.5-2.5 fold, and 3.5-4 fold, respectively, relative to the expression of the markers in exosomes derived from mesenchymal stem cells. In some embodiments, the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 are expressed in the induced extracellular vesicles in an amount of 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold, respectively, relative to the expression of the marker in exosomes derived from mesenchymal stem cells. The induced extracellular vesicles of the invention are substantially different from exosomes, for example, the induced extracellular vesicles IEVs of the invention highly express Syntaxin 4, and the expression levels of Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5 are significantly higher than those of exosomes (see example 3). In addition to differential marker expression, the induced extracellular vesicles IEVs also exhibit characteristics that are distinct from stem cells and other extracellular vesicles such as exosomes, either functionally or therapeutically. For example, IEVs significantly shorten the clotting time of most plasma in vitro, and the procoagulant effect is better than that of exosomes (see test example 2). For example, the mechanism of IEVs in treating hemophilia mice is independent of PS and TF, whereas in previous literature reports, the promotion of coagulation by extracellular vesicles was highly dependent on both PS and TF on their surface (see test example 2). In some embodiments, the inducing extracellular vesicles further express CD29, CD44, CD73, CD 166; and does not express CD34, CD 45. In some embodiments, the inducing extracellular vesicles further express one or more of CD9, CD63, CD81, and C1 q.

In some embodiments, the formulation is an injection, an oral formulation, or a topical formulation.

In some embodiments, the formulation is an injection.

In some embodiments, the formulation is an intravenous, intramuscular, subcutaneous, or intrathecal injection.

In some embodiments, the formulation further comprises a pharmaceutically acceptable carrier.

In some embodiments, the formulation vehicle comprises one or more of diluents, excipients, fillers, binders, disintegrants, surfactants, and lubricants.

In some embodiments, there is also provided a method of preparing the induced extracellular vesicles, comprising the steps of:

1) culturing mesenchymal stem cells in vitro, and washing with PBS for 2-5 times when the cells are 80% -90% confluent;

2) adding the mesenchymal stem cells prepared in the step 1) into a serum-free culture medium containing 500-1000nM staurosporine, incubating for 16-24h at 37 ℃, and collecting cell supernatant;

3) centrifuging the cell supernatant collected in step 2) at 500-;

4) centrifuging the cell supernatant collected in step 3) at 1500-;

5) centrifuging the cell supernatant collected in the step 4) at 10000-;

preferably, a washing step of the induced extracellular vesicles is also included;

preferably, the cleaning step specifically comprises: 6) resuspending the induced extracellular vesicles prepared in step 5) with PBS, and centrifuging at 10000-30000g at 4 ℃ for 15-60 minutes to obtain precipitates which are the induced extracellular vesicles.

In the present invention, the "mesenchymal stem cell" refers to a pluripotent stem cell having all the commonalities of stem cells, i.e., self-renewal and multipotential differentiation ability. The mesenchymal stem cells can be derived from bone marrow, fat, blood (e.g., extrinsic blood), synovium, bone, muscle, lung, liver, pancreas, oral cavity, craniomaxillofacial (e.g., deciduous teeth, dental pulp, periodontal ligament, gingiva, apical tooth papilla, etc.), and amniotic fluid, umbilical cord, i.e., the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, skeletal mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreas mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.

In the present invention, the "mammal" is selected from a mouse, rat, dog, cat, rabbit, monkey or human.

In the present invention, the term "preventing" means that when used in a disease or condition, the agent reduces the frequency of, or delays the onset of, symptoms of the medical condition in a subject as compared to a subject to which the agent is not administered.

In the present invention, the term "treating" refers to alleviating, alleviating or ameliorating the symptoms of a disease or disorder, ameliorating the underlying metabolic-induced symptoms, inhibiting a disease or symptom, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, causing regression of a disease or disorder, alleviating a condition caused by a disease or disorder, or arresting the symptoms of a disease or disorder.

Drawings

FIG. 1 is a flow cytometric assay of example 1.

Figure 2 is a technical scheme for preparing IEVs in example 2.

FIG. 3 shows MSCs (10) analyzed by flow cytometry⁶Individual MSCs) produced statistics of the number of IEVs.

FIGS. 4A-4F are diameter measurements of IEVs particles: FIG. 4A is a particle diameter distribution plot for flow-assay IEVs; FIG. 4B is a side scatter light (SSC) analysis of the scattered light intensity of IEVs showing the distribution of IEVs particle diameter; FIG. 4C is a graph of the intensity of light scattered from IEVs analyzed using standardized small particle microspheres from Bangs Laboratories, Inc., showing the particle diameter distribution of IEVs; FIG. 4D is Transmission Electron Microscopy (TEM) observed IEVs, showing the particle diameter distribution of the IEVs; FIG. 4E is Nanoparticle Tracking Analysis (NTA) showing the distribution of IEVs particle diameter; fig. 4F shows the particle size distribution of IEVs at the level of single vesicles using nano-flow detection technique.

FIGS. 5A-5K are results of analysis of surface membrane proteins of IEVs by flow cytometry.

FIGS. 6A-6D are content analyses of IEVs: FIG. 6A shows the results of quantitative analysis of proteomics of MSCs, MSCs-Exosomes and MSCs-IEVs by DIA quantification technique; FIG. 6B is a heat map drawn screening for IEVs specific highly expressed proteins; FIG. 6C is the results of GO enrichment analysis of differential proteins for IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 molecules; FIG. 6D is a result of verifying that MSCs, MSCs-Exosomes, MSCs-IEVs express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 by western blot.

FIG. 7 is a Kaplan-Meier survival curve showing that IEVs injection significantly extended mouse life.

Figure 8 shows that multiple injections of BMMSCs-derived IEVs significantly reduced body weight and hair loss in aged mice.

Figure 9 is a significant reduction in spleen volume and weight in aged mice given multiple injections of BMMSCs-derived IEVs.

Fig. 10A is a micct image of different treatment groups injected multiple times with IEVs from BMMSCs sources.

Figure 10B is a statistical analysis showing that multiple injections of MSCs-derived IEVs significantly increased bone density and bone mass counts in aged mice.

FIG. 11A shows the statistical analysis of CFU-F and BrdU stained toluidine blue, BrdU, for different treatments.

Fig. 11B is a graph showing the ability of BMMSCs to form mineralized nodules under different treatments with alizarin red staining, and oil red O staining showing the number of adipocytes in MSCs.

Figure 12A is a schematic representation of the dynamic metabolism of IEV at the skin surface in example 10.

Figure 12B shows that over time, IEV gradually moved from the subcutaneous tissue to the dermal layer and epidermis.

FIG. 12C shows that PKH26-IEV was found in hair follicles found in hairs pulled from mouse bodies at day 7.

Figure 13A shows the distribution of IEV throughout the body of mice on

days

1, 3, and 7 as measured by in vivo imaging techniques.

FIG. 13B shows distribution of IEV in each mouse organ as measured by in vivo imaging technique.

FIG. 13C is a comparison of the distribution of IEV in the organs of the mice at day 7 with that of the control group.

FIG. 13D is a schematic representation of the dynamic metabolism of IEV in the colon on

days

1, 3, and 7; wherein M represents the muscularis mucosae layer and V represents the chorion.

Figure 13E shows a schematic representation of the metabolism of IEV in the nail and incisors of mice at day 3.

Fig. 14A is a graph showing the regeneration of back hair on

days

0, 10, and 14 for the mice treated differently in example 11.

FIG. 14B is a graph showing statistical analysis of the area of regenerated back hair on

days

0, 10 and 14 for the mice treated differently in example 11.

Figure 15 shows the wound healing promoting effect of IEV and MSC treatment in example 12.

Figure 16 is IEVs treatment of sjogren's syndrome: ievs treatment of the effects of sjogren's syndrome (sjogren's syndrome) salivary flow rate; staining results of IEVs in treating Sjogren syndrome submandibular gland HE; C. treatment of the effects of sjogren's syndrome on B cells.

Figure 17 is an in vivo procoagulant effect of IEVs in hemophilia a mice.

Fig. 18A-18D are graphs showing the change in levels of various clotting factors following injection of IEVs into hemophilia a mice: FIG. 18A is a variation of factor VIII; FIG. 18B shows the change in vWF factor; FIG. 18C shows changes in Tissue Factor (TF); FIG. 18D shows the prothrombin profile.

Fig. 19A-19B are graphs showing the effect of blockade of PS and TF, respectively, on the in vivo therapeutic efficacy of IEVs in a mouse model of hemophilia a.

FIG. 20 is a graph comparing the therapeutic effect of IEVs and Exosomes from the same MSCs on hemophilia A mice.

Note: in the figures, WT is a wild-type mouse; the HA group is a hemophilia a mouse model; HA + IEVs treatment with IEVs for hemophilia a mouse model; HA + PS-IEVs PS-negative IEVs administered to hemophilia A mouse model; HA + TF-IEVs to TF-negative IEVs for hemophilia A mouse model; HA + Exosomes subjects haemophilia a mouse models were given Exosomes treatment.

Detailed Description

The technical solutions of the present application are further illustrated by the following specific examples, which do not represent a limitation to the scope of the present application. Insubstantial modifications and adaptations of the concepts taught herein by others are intended to be covered by the present disclosure.

STS in the present invention is staurosporine.

IEVs in the present embodiment are simply referred to as inductive vesicles, which may also be referred to as Inductive Extracellular Vesicles (IEVs). Induced extracellular vesicles are a type of subcellular product produced by a precursor cell (e.g., a stem cell, such as a mesenchymal stem cell, among others) that is intervened or induced to undergo apoptosis during its normal survival. Preferably, the precursor cells are early precursor cells. Usually this class of subcellular products, with membrane structures, express apoptotic markers, and contain, in part, genetic material DNA. The inventors have found that induced extracellular vesicles are a class of substances that is distinguished from cells and conventional extracellular vesicles (e.g., exosomes, etc.).

Normal Extracellular Vesicles (EVs) refer to small membrane-structured bodies of nanometer size that are spontaneously secreted by cells during normal culture processes or under in vivo physiological conditions, have diameters varying from 40 to 1000nm, are mainly composed of Microvesicles (MVs) and Exosomes (Exosomes), and contain various signal molecules such as RNA and proteins.

"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present application.

The term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and/or" means that any one of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, combination, construction, etc., is described as comprising (or comprising) components a, B, C, and/or D, the composition can comprise a alone; b alone; separately comprises C; separately contain D; a combination comprising A and B; a combination comprising A and C; a combination comprising A and D; a combination comprising B and C; a combination comprising B and D; a combination comprising C and D; a combination comprising A, B and C; comprising a combination of A, B and D; a combination comprising A, C and D; comprises a combination of B, C and D; or A, B, C and D are used in combination.

EXAMPLE 1 isolation and culture of BMMSCs

Excess CO was used according to the guidance of the animal ethics Committee₂The mice are sacrificed, the tibia and the femur are taken off under the aseptic condition, the muscle and connective tissue attached to the tibia and the femur are stripped, the metaphysis is further separated, the marrow cavity is exposed, PBS with the volume fraction of 10% fetal calf serum is extracted by a 10mL aseptic syringe to repeatedly wash the marrow cavity, after filtration by a 70-micron pore cell filter screen, 500g is centrifuged for 5min, the cell sediment at the bottom is collected after the supernatant is removed, PBS is resuspended, the PBS is centrifuged again for 5min by 500g, and the final cell sediment is collected. The cells were then flow sorted and Bone Marrow Mesenchymal Stem Cells (BMMSCs) were sorted using CD 34-and CD90+ as sorting criteria. Finally, cells were resuspended in Dex (-) medium and seeded on 10cm diameter cell culture dishes at 37 ℃ with 5% CO₂And (5) culturing. After 24h, the non-adherent cells in the supernatant were aspirated off, washed with PBS, and then cultured by adding Dex (-) culture medium. After 1 week, an equal amount of Dex (+) medium was added, and after 1 week, dense colonies of primary BMMSCs were observed. BMMSCs were digested by trypsin incubation at 37 ℃ and expanded by passage, after which Dex (+) culture medium was changed every 3 days and passaged after confluency. Subsequent experiments were performed using P2 generation BMMSCs.

The components of the Dex (-) culture medium are shown in Table 1, and the components of the Dex (+) culture medium are shown in Table 2:

TABLE 1 Dex (-) composition of culture solution

TABLE 2 Dex (+) culture solution formulation Table

The purity of the isolated BMMSCs was assessed by flow cytometry analysis of surface markers. For surface marker identification, after collection of P2 generation BMMSCs by trypsinization, the BMS was washed 1 time with PBS at 5X 10⁵Resuspend cells at density/mL in PBS containing 3% FBS, add 1 μ L PE fluorescently conjugated CD29, CD44, CD90, CD45 and CD34 antibodies, and blank group does not. Incubating at 4 ℃ in dark for 30min, washing for 2 times by PBS, and detecting on a machine. The results of the detection are shown in FIG. 1.

Example 2 preparation of BMMSC-derived IEV

MSCs (bone marrow-derived MSCs) cultured up to passage 2 in example 1 were cultured in the medium (Dex (+) culture) of example 1 until the cells reached 80% -90% confluence, washed 2 times with PBS, added with 500nM STS-containing serum-free medium (500 nM STS was added to the medium of example 1), incubated at 37 ℃ for 16-24h, cell supernatant was collected, centrifuged at 4 ℃ at 800g for 10 min, supernatant was collected, centrifuged at 4 ℃ at 2000g for 10 min, collected at 4 ℃ at 16000g for 30min, and the resulting precipitate was IEV. 500 μ l PBS heavy suspension precipitation, 4 degrees C under 16000g centrifugation for 30 minutes, get the washing IEV.

The preparation route is shown in figure 2.

Comparative example 1 exosome isolation and extraction from homogeneous MSC

MSCs (bone marrow-derived MSCs, BMMSCs) cultured up to passage 2 in example 1 were cultured with the medium in example 1, washed 2 times with PBS when the cells were confluent by 80% to 90%, serum-free medium was added, incubated at 37 ℃ for 48 hours, and cell supernatant was collected for isolation and extraction of exosomes.

The extraction step comprises: centrifugation at 800g for 10 min-collection of supernatant-centrifugation at 2000g for 10 min-collection of supernatant-centrifugation at 16000g for 30 min-collection of supernatant-centrifugation at 120000g for 90 min-removal of supernatant, resuspension of pellet with sterile PBS at 120000g for another 90 min, removal of supernatant, collection of bottom exosomes, and resuspension with sterile PBS.

Example 3 analysis of IEVs

(1) Quantification of IEVs and analysis of Membrane proteins

Quantitative analysis of the IEVs obtained in example 2 was carried out by flow cytometry at the

measurement time points

1h, 4h, 8h, 16h and 24h, and the results showed 10⁶The MSCs can respectively produce 0.76 multiplied by 10 after induction to 1h, 4h, 8h, 16h and 24h⁸1.29X 10⁸1.95 × 10⁸2.48 x 10⁸3.14 x 10⁸From the IEVs, it can be seen that after 24h induction, a single MSC can yield 300 IEVs (fig. 3).

Furthermore, flow assays found that the particle diameter distribution of the IEVs was concentrated at below 1 μm, representing 94.97% (FIG. 4A).

Side scatter light (SSC) analysis also showed IEVs scattered light intensity concentrated in the range below 1 μm (FIG. 4B).

Further, the scattering intensity of IEVs was analyzed by standardized small particle microspheres (0.2 μm, 0.5 μm, 1 μm) manufactured by Bangs Laboratories, Inc., and the results showed that the particle diameters of IEVs were all below 0.2 μm (FIG. 4C).

The results of Transmission Electron Microscopy (TEM) were similar to flow measurements, with most vesicles at 200nm and below 200nm in diameter (fig. 4D).

The Nanoparticle Tracking Analysis (NTA) results were consistent with transmission electron microscopy observations, with IEVs having an average particle diameter of 169nm (FIG. 4E).

Particle size measurements at the level of single vesicles were performed using state-of-the-art nano-flow detection techniques and also showed that the average particle diameter of the IEVs was 100.63 nm (fig. 4F).

Analysis of the surface membrane proteins of the IEVs extracted in example 3 using flow cytometry showed that the iecs from MSCs were able to express similar surface proteins as MSCs, i.e. CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. Meanwhile, IEVs were able to express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (see fig. 5A-5K).

(2) Content analysis of IEVs

Proteomic quantitative analysis of MSCs, MSCs-Exosomes (extracted in comparative example 1), MSCs-IEVs (obtained in example 2) was performed using protein DIA quantification technique. The results showed that the protein content expression of MSCs-Exosomes and MSCs-IEVs had higher overlap with the mother cells, and 170 proteins were specifically highly expressed in IEVs (fig. 6A). By bioinformatics analysis, the specific high-expression protein of IEVs is screened, a heat map is drawn (FIG. 6B), and further combined with the GO enrichment analysis result of differential protein, the specific high-expression molecules of Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 of IEVs are confirmed (FIG. 6C). Compared with Exosomes from the same MSCs, the expression levels of 5 characteristic molecules of IEVs are all significantly up-regulated, specifically: the expression levels of markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntaxin 4 in IEVs were 1.76-fold, 2.81-fold, 2.41-fold, 3.68-fold and 4.45-fold, respectively, relative to the corresponding markers in exosomes. Finally, the western blot technique is used to perform the verification again, and the result is consistent with the quantitative analysis result of DIA (FIG. 6D).

MSCs-Exosomes: refers to exosomes derived from MSCs.

MSCs-IEVs: refer to IEVs derived from MSCs.

Wherein the MSCs in the assay of the contents are of the same cell line as the MSCs from which the exosomes and IEVs are extracted.

General Experimental methods for examples 4-9

1. Experimental Material

60 normal wild type C57 mice were divided into 3 groups, i.e., Control group (Control), IEV group, MSC group, 20 mice per group.

IEV was prepared for example 2 and MSC was BMMSC prepared in example 1.

2. Experimental methods

Three groups of mice were injected with PBS, IEV (prepared in example 2), MSC (prepared in example 1) in tail vein, once a month from 8 months of age. Wherein the dose of IEV is 1 × 10 per mouse⁶Resuspending each IEVs in 200 μ L PBS, mixing, standing on ice, and injecting via tail vein within 30 min; MSC group mice were injected with the same amount of cells (1X 10) per injection⁶Individual MSCs); control group mice were injected with an equal volume of solvent (PBS,200 ul/mouse).

Example 4 multiple injections of BMMSCs-derived IEVs significantly extended mouse lifespan

Groups of mice were prepared using the general experimental procedure described above.

Mice status and death status were monitored continuously. And subjected to Kaplan-Meier survival analysis. Where the control and treatment groups were analyzed for statistical differences using the log rank test, P values less than 0.05 were considered statistically different.

The results of this example show that both IEVs and MSC injection can improve survival rate of aged mice, and Kaplan-Meier survival analysis shows that both IEV group and MSC group significantly prolonged the lifespan of mice compared to the control group (fig. 7).

Example 5 multiple injections of BMMSCs-derived IEVs significantly reduced the body weight of aged mice

The mice of each group were weighed at 24 months of age and statistically analyzed. The results of this example show that both IEV injection and MSC injection significantly reduced the body weight of the aged mice (FIG. 8A).

Example 6 multiple injections of BMMSCs-derived IEVs significantly reduced the appearance of hair loss in aged mice

The hair status was recorded by photographing the mice at 24 months of age, and the photographs showed that IEV injection significantly reduced the phenomenon of mouse depilation (fig. 8B). Compared to the treatment group injected with MSC, the treatment group injected with IEV had almost complete recovery of hair loss. The depilating was not significantly improved in the MSC-injected group.

Example 7 multiple injections of BMMSCs-derived IEVs significantly reduced spleen volume and weight in aged mice

At 24 months of age, 5 mice were randomly selected per group, and their spleens were taken after sacrifice, photographed and weighed. The results show that IEV injection significantly reduced mouse spleen volume and weight (figure 9). However, the mice injected with MSC had spleen with less reduction in volume and weight than the treatment group injected with IEV.

Example 8 multiple injections of BMMSCs-derived IEVs significantly enhanced bone density in aged mice

At 24 months of age, randomly taking 5 mice from each group, taking femurs of the mice after sacrifice, and analyzing bone mineral density and bone volume related indexes such as BMD, BV/TV and the like by using a micro CT.

And (3) MicroCT analysis: after fixation of mouse femurs in 4% PFA, femurs were analyzed using a high resolution Scanco MicroCT50 scanner (Scanco Medical AG). The samples were scanned at 20kVp and 200 μ A using a voxel size of 20 μm. The data set was analyzed using Amira 5.3.1 software (Visage Imaging) to reconstruct the image and measure bone mineral density.

The results showed that both IEV and MSC injections enhanced bone mineral density BMD and bone mass number BV/TV in aged mice (FIGS. 10A, 10B).

Example 9 multiple injections of BMMSCs-derived IEVs significantly enhanced BMMSCs function in aged mice

At 24 months of age, 5 mice per group were randomly selected and their BMMSCs were isolated and cultured.

Isolation of BMMSCs: bone marrow-derived all nucleated cells (ANCs, 15X 10) from femurs⁶) The single suspension was seeded in 100mm petri dishes (Corning) and incubated with 5% CO at 37 deg.C₂And (4) incubation. After 24 hours, non-adherent cells were removed and placed in α minimal essential medium (α -MEM, Invitrogen) supplemented with 20% Fetal Bovine Serum (FBS), 2mM L-glutamineAdherent cells were cultured for an additional 14 days (Invitrogen), 55. mu.M 2-mercaptoethanol (Invitrogen), 100U/ml penicillin and 100. mu.g/ml streptomycin (Invitrogen). These adherent single colonies were passaged at passage 1 with frequent medium changes to eliminate potential hematopoietic cell contamination. Determination of colony forming units-fibroblasts (CFU-F): will be 1 × 10⁶Single suspension BMMSCs were plated in 60mm dishes (Corning). After 16 days, the cultures were washed with PBS and stained with 1% toluidine blue solution containing 2% paraformaldehyde (PFA, Sigma-Aldrich). The cell clusters were counted under a microscope and cell clusters with more than 50 cells were considered as colonies.

Cell proliferation assay: proliferation of BMMSCs was assessed using a bromodeoxyuridine (BrdU) incorporation assay. Briefly, BMMSCs were administered at 1X 10 per well⁴Individual cells were seeded in 12-well plates for 24 hours, and the cultures were incubated with BrdU solution (1:100) (Invitrogen) for 24 hours and stained with BrdU staining kit (Invitrogen) according to the manufacturer's instructions. BrdU positive and total cell counts were counted in 10 images per sample. The number of BrdU positive cells was expressed as a percentage of the total number of cells. For each experimental group, the BrdU assay was repeated with five independent samples.

In vitro osteogenic differentiation: BMMSCs were cultured under osteogenic induction conditions, including 2m M β -glycerophosphate (Sigma-Aldrich), 100 μ ML-ascorbic acid, 2-phosphoric acid (Wako), and 10nM dexamethasone (Sigma-Aldrich) in growth medium. After 3 weeks of induction, matrix mineralization was detected by 1% alizarin red S (Sigma-Aldrich) staining and the positive area of staining was quantified using NIH ImageJ software and shown as a percentage of the total area.

In vitro adipogenic differentiation: for adipogenesis induction, 500nM isobutylmethylxanthine (Sigma-Aldrich), 60. mu.M indomethacin (Sigma-Aldrich), 500nM hydrocortisone (Sigma-Aldrich), 10. mu.g/ml with insulin (Sigma-Aldrich) and 100nM L-ascorbyl phosphate. This was added to conventional mouse BMMSC growth medium. After 7 days of induction, the cultured cells were stained with oil red O (Sigma-Aldrich) and positive cells were quantified under a microscope and shown as the number of positive cells as a percentage of total cells.

The results show that 24-month old mice have reduced MSC self-renewal capacity, including colony formation (CFU-F colony forming unit fibroblasts), and proliferation capacity (BrdU bromodeoxyuridine) relative to 6-month old mice; the osteogenic ability is reduced and the fat formation is increased. These all reflect impaired MSC capacity, whereas IEV-injected mice had significantly improved MSC function relative to the non-injected group (fig. 11A, 11B).

Example 10 IEV can drain through the skin and hair

Take 4X 10⁶The IEV prepared in example 2 was re-suspended with DIR, 200. mu.l PBS, and systemically injected into the nude mice BALB/C-nu/nu via the caudal vein, and the distribution of IEV on the skin surface was observed after 1, 3, and 7 days using a living body imaging device, and the results are shown in FIGS. 12A-12C.

Fig. 12A shows that IEV can reach the skin surface, at the most by day 3, and at day 7, disappear substantially, showing the dynamic metabolic processes of IEV at the skin surface (fig. 12A). Immunofluorescence results show that PKH26-IEV systemically injected C57 mice gradually moved from subcutaneous tissue to dermal and epidermal layers over time. The presence of a large number of IEVs was observed on the stratum corneum layer on the skin surface at day 7, suggesting that the systemically injected IEVs could be excreted as the stratum corneum of the skin sloughs (fig. 12B). Meanwhile, the presence of PKH26-IEV in the hair follicles was found in the hairs pulled down from the mouse body surface on day 7, suggesting that the IEV injected systemically could also be metabolized out as the hairs are shed (fig. 12C).

In addition, both in vivo imaging data and immunofluorescence results showed that IEVs are distributed in large numbers in internal organs such as liver, spleen, bone marrow, lymph, etc., and not in heart, kidney, brain, but in colon, teeth and nails, further demonstrating that IEVs are excreted in vitro with metabolism (fig. 13A-13E).

This example shows that IEV can be expelled through the skin and hair, indicating that it is safe to inject or increase the level of IEV in the body.

Example 11 IEV is able to promote Hair regrowth

Female C57 mice (hair quiescent) at 7 weeks were depilatory treated with PBS, IEVs, MSC and 2% Minoxidil (Minoxidil) s.c. injection, respectively. Comparing the area of hair regrowth on the backs of mice on day 10 and day 14, the experimental results showed that IEVs and MSCs had a significant effect of promoting hair regrowth compared to the control group, and both IEVs and MSCs had a more significant effect of promoting hair regrowth on day 14 than the conventional alopecia-preventing drug minoxidil (fig. 14A-14B).

Example 12 IEVs promote wound healing

1cm × 1cm of full-thickness skin wounds were made in mice, and the promotion of wound healing by systemic injection of MSCs and IEVs was examined. The results showed that both IEVs and MSCs had a promoting effect on wound healing (fig. 15), with no significant difference.

Combined with examples 10, 11, 12, metabolic studies of IEV have shown that it is excreted through the skin and skin appendages and has a positive effect on these tissues.

Test example 1

(1) The detection step or method comprises: taking 8-week-old Sjogren Syndrome (SS) model mice, injecting MSCs and IEVs through a tail vein system, taking materials 4 weeks after injection, detecting the flow rate of saliva, collecting salivary gland samples, and performing paraffin section HE staining and B cell marker B220 staining.

(2) As a result: as shown in fig. 16A-fig. 16C, the results showed that, compared to the effect of the mice bone marrow mesenchymal stem cells and their derived IEVs on the salivary flow rate of sjogren's syndrome, there was a slight recovery of salivary flow rate after mesenchymal stem cell treatment, and no improvement in salivary flow rate after IEVs treatment was seen (. # p <0.05 compared to WT group, # p <0.001 compared to MSCs group). IEVs injection did not alter inflammatory infiltration of salivary glands and B cell accumulation.

Test example 2

The IEVs obtained in example 2 and the Exosomes extracted in comparative example 1 were tested for their in vitro procoagulant effects using an in vitro clotting assay. The results are shown in Table 3, where IEVs significantly reduced the in vitro clotting time of most plasma, and the procoagulant effect was better than that of Exosomes.

However, for plasma deficient in factors II, V, X, IEVs failed to exert in vitro procoagulant effects, suggesting that the in vitro procoagulant effects of IEVs are more focused upstream of the common pathway of coagulation.

TABLE 3

Hemophilia A mice (factor VIII deficient) were used as a model and injected by tail vein with 9X 10⁸IEVs, observed for in vivo procoagulant effects of IEVs. The results are shown in fig. 17, which shows that after IEVs treatment, the bleeding tendency of hemophilia mice can be significantly improved, and the treatment effect can be sustained and stably maintained for 14 days.

Experimental results show that IEVs are able to exert a significant procoagulant effect in vitro. And can remarkably improve bleeding tendency after in vivo injection, and can be used for improving bleeding tendency caused by hemophilia A. The levels of various coagulation factors in the plasma of mice were also measured, and no significant change was observed in coagulation factor VIII, vWF factor, Tissue Factor (TF), and prothrombin (fig. 18A, 18B, 18C, 18D).

In hemophilia a mouse model, normal IEVs, PS negative IEVs and TF negative IEVs were injected, respectively, and after 7 days, tail-clipping experiments were performed, and the results are shown in fig. 19A and 19B, where the blockade of PS and TF did not affect the in vivo therapeutic effect of IEVs, which primarily indicates that the mechanism of IEVs in treating hemophilia mice is independent of PS and TF. In the past literature reports, the coagulation promoting effect of extracellular vesicles is highly dependent on PS and TF on the surface of the extracellular vesicles, and the in vivo experimental results of IEVs are inconsistent with the previous researches, which suggests that the IEVs may have a new action mechanism to exert the coagulation promoting effect under the in vivo environment.

For hemophilia A mouse model, injection treatments (9X 10) of IEVs (obtained in example 2) and Exosomes (extracted in comparative example 1) from the same MSCs were performed, respectively⁸One), the results show that IEVs are able to significantly correct bleeding tendencies in mice, while Exosomes have no significant therapeutic effect (fig. 20).

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the application and should not be taken as limiting the scope of the application. Rather, the scope of the application is defined by the appended claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. Use of induced extracellular vesicles for the preparation of a formulation for prolonging the lifespan of a mammal or for treating or preventing aging.

2. The use of claim 1, wherein the induced extracellular vesicles achieve longevity prolongation or treatment or prevention of aging in a mammal by restoring proliferation and/or differentiation of damaged cells.

3. The use of claim 1, wherein the formulation is a formulation for the treatment or prevention of geriatric obesity;

preferably, the preparation is a preparation for treating or preventing alopecia due to aging;

preferably, the preparation is an agent for treating or preventing splenomegaly;

preferably, the formulation is a formulation for treating or preventing osteoporosis, bone loss or bone aging;

preferably, the preparation is selected from a pharmaceutical preparation or a health product preparation.

4. The use of claim 1, wherein the inducing extracellular vesicles are for use in reducing the weight of an elderly individual;

preferably, the induced extracellular vesicles are used to reduce alopecia senilis;

preferably, the induced extracellular vesicles are used to reduce spleen weight or volume;

preferably, the induced extracellular vesicles are used to increase bone density;

preferably, the induced extracellular vesicles are used to increase bone volume fraction.

5. The application of the induced extracellular vesicles in preparing a preparation for treating or preventing senile alopecia; preferably, the preparation is selected from a pharmaceutical preparation or a health product preparation.

6. Use of induced extracellular vesicles for the preparation of an anti-ageing, and/or repairing, and/or regenerating preparation of the skin and/or skin appendages;

preferably, the skin is epidermis, dermis, or subcutaneous tissue;

preferably, the skin appendages are hair, sweat glands, sebaceous glands, nails, or toenails;

preferably, the preparation is an agent for treating or preventing alopecia, or an agent for promoting hair regeneration;

preferably, the formulation is one that promotes the repair and/or regeneration of a skin wound or scar;

7. The use of any one of claims 1 or 5 or 6, wherein the induced extracellular vesicles are produced from stem cells selected from the group consisting of mesenchymal stem cells or induced pluripotent stem cells;

preferably, the mesenchymal stem cells are selected from one or more of blood mesenchymal stem cells, bone marrow mesenchymal stem cells, urine mesenchymal stem cells, oral mesenchymal stem cells, adipose mesenchymal stem cells, placenta mesenchymal stem cells, umbilical cord mesenchymal stem cells, periosteal mesenchymal stem cells and skin mesenchymal stem cells;

preferably, the stem cell is selected from one or more of induced pluripotent stem cell, blood mesenchymal stem cell, bone marrow mesenchymal stem cell, adipose mesenchymal stem cell, umbilical cord mesenchymal stem cell, oral mesenchymal stem cell and skin mesenchymal stem cell;

preferably, the stem cells are selected from one or more of induced pluripotent stem cells, blood stem cells or bone marrow mesenchymal stem cells.

8. The use of any one of claims 1, 5 or 6, wherein the induced extracellular vesicles are induced by the addition of staurosporine, uv irradiation, starvation, or thermal stress or a combination thereof to induce apoptotic production of mesenchymal stem cells;

preferably, the stem cell extracellular vesicles are produced by inducing apoptosis of mesenchymal stem cells by adding staurosporine.

9. The use according to claim 8, characterized in that the concentration of staurosporine is greater than or equal to 1 nM;

preferably, it is 1-15000 nM;

preferably 200-; preferably, 250-1000 nM; preferably 500-1000 nM.

10. The use of any one of claims 1 or 5 or 6, wherein the inducing extracellular vesicles have a diameter of 0.45 μm or less;

preferably, the diameter of the inducing extracellular vesicles is 0.05-0.45 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.1-0.45 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.1-0.35 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.35 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.3 μm;

preferably, the diameter of the inducing extracellular vesicles is 0.15-0.2 μm.