WO2025067020A1 - Use of adult-stem-cell-derived intracellular nanovesicle in anti-aging and hair follicle regeneration - Google Patents

Abstract

The use of an adult-stem-cell-derived intracellular nanovesicle in the anti-aging, repair and regeneration of the skin and/or cutaneous appendages, especially the use of a mesenchymal-stem-cell-derived intracellular nanovesicle in the anti-aging and hair follicle regeneration of the skin. Such intracellular nanovesicle can reduce the expression of aging-related molecules, improve the proliferation activity of human skin fibroblasts, improve SOD activity, effectively improve cell aging (including endogenous aging (natural aging) and exogenous aging (such as photoaging)), and can also effectively improve aging in an animal model. In addition, the intracellular nanovesicle can also promote hair follicle development and regeneration, facilitate the regeneration of more hair, and is expected to be used for preventing and treating alopecia. Therefore, the intracellular nanovesicle has very good application prospects and research value.

Description

Application of intracellular nanovesicles derived from adult stem cells in anti-aging and hair follicle regeneration Technical Field

The present invention relates to the field of medical cosmetology technology, and specifically to an application of intracellular nanovesicles derived from adult stem cells in the anti-aging, repair and regeneration of skin and/or skin appendages, in particular to the application of intracellular nanovesicles derived from mesenchymal stem cells in the anti-aging of skin and hair follicle regeneration.

Background Art

Skin aging is the result of the combined effects of genes and external environmental factors. Skin aging is divided into endogenous aging and exogenous aging according to different influencing factors. Endogenous aging refers to the natural aging that occurs over time at the genetic level. Exogenous aging refers to the accumulation of skin damage caused by environmental factors such as ultraviolet radiation, smoking, and toxic chemicals. Skin photoaging is the most important exogenous aging, which manifests as rough, thickened, loose, and wrinkled skin, localized excessive pigmentation or capillary dilation, and may even lead to various benign or malignant tumors (such as solar keratosis, squamous cell carcinoma, malignant melanoma, etc.).

Skin aging has a great impact on people's pursuit of "beauty". Modern research shows that skin aging not only affects the structure and function of each layer of skin, but also promotes the aging of other organs in the body. Therefore, the importance of research on anti-skin aging is self-evident. Research on anti-skin aging can not only help restore the youthful vitality of aging skin, improve the psychological state and quality of life of aging individuals, but also delay the aging process of the whole body and reduce potential health risks.

Hair loss is also a common and frequently occurring disease in clinical dermatology, including senile alopecia, alopecia areata, chemotherapy-induced alopecia, and seborrheic alopecia, among which most patients suffer from alopecia areata or seborrheic alopecia. Currently, commonly used drugs for hair loss treatment include retinoic acid, atropine, glucocorticoids, etc. However, long-term use of Western medicines can cause significant side effects.

Exosomes are small extracellular vesicles with a diameter of 40-150nm and a double-layer phospholipid membrane structure secreted by cells. However, compared with other treatment methods, the production and extraction costs of exosomes are relatively high, and there are certain technical difficulties, which limit the feasibility of their large-scale application. In fact, there are a large number of nanovesicles in cells, which are free between various membrane-rich organelles, mediating the intracellular material transport and the cell's secretory pathway. Among them, constitutive secretory vesicles are the main type of intracellular vesicles (IVs), which are produced by the endoplasmic reticulum and exchange substances with the Golgi apparatus, lysosomes and cell membranes, mediating the production, transportation and secretion of various secretory proteins in cells. It is rich in content and contains a large number of secretory proteins, lipids and nucleic acid molecules. In addition, our previous results found that compared with EVs, these intracellular nanovesicles are smaller in size than exosomes, have a higher yield, and contain more proteins, lipids and nucleic acids. Therefore, the use of intracellular nanovesicles for anti-aging and hair follicle regeneration research and the search for new anti-aging and hair follicle regeneration drugs have great application prospects.

Summary of the invention

To overcome the deficiencies of the prior art, the present invention provides an application of intracellular nanovesicles derived from adult stem cells (particularly mesenchymal stem cells) in the anti-aging, repair and regeneration of skin and/or skin appendages.

In a first aspect of the present invention, there is provided a use of intracellular nanovesicles derived from adult stem cells (particularly mesenchymal stem cells) in the preparation of anti-aging and photoaging skin repair products.

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

Specifically, the vesicles are round in shape.

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

Specifically, the vesicles lowly express exosome markers, and highly express marker proteins of intracellular membrane-rich organelles and the Clathrin protein family.

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

Specifically, the vesicles are prepared by a method comprising an ultrasonic treatment step. More specifically, the vesicles are prepared by a method comprising an ultrasonic treatment, a centrifugation treatment, and an ultracentrifugation treatment step (performed sequentially).

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

(1) dispersing mesenchymal stem cells in a suspension solvent and subjecting them to ultrasonic treatment;

(2) centrifuging the liquid obtained in step (1) once or multiple times to obtain the supernatant;

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

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

Specifically, the cells in step (1) are cells separated after culture, digestion and washing (after discarding the cell culture medium, digestion and washing). The possibility of isolating extracellular vesicles can be ruled out by steps such as lysis and washing).

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

Specifically, the cell density of the cells in the suspension solvent is 1-4×10 ⁶ cells/mL, such as 1×10 ⁶ cells/mL, 2×10 ⁶ cells/mL, 3×10 ⁶ cells/mL, 4×10 ⁶ cells/mL. In some embodiments of the present invention, the cell density is 1×10 ⁶ cells/mL.

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

Specifically, the adult stem cells refer to undifferentiated cells existing in a differentiated tissue, which can self-renew and specialize to form cells that constitute this type of tissue. Under certain conditions, adult stem cells either produce new stem cells or differentiate according to a certain procedure to form new functional cells, thereby maintaining a dynamic balance of growth and decline of tissues and organs. For example, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells (NSC), etc., especially mesenchymal stem cells.

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

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

Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-35% (e.g., 20%, 22%, 24%, 25%, 30%, 35%); in some embodiments of the present invention, the amplitude of the ultrasonic treatment is 20%.

Specifically, the time of ultrasonic treatment in step (1) is 15-60s (e.g., 15, 20, 25, 30, 40, 50, 60s), preferably 15s; in some embodiments of the present invention, the time of ultrasonic treatment is 15s, on 2s, off 2s.

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

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

10000-30000 g (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 2000 g for 10 minutes.

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

Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100,000-180,000 g (e.g., 100,000, 120,000, 140,000, 150,000, 160,000, 180,000 g), 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 150000 g 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 present invention, the resuspension solvent is PBS.

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

In some embodiments of the present invention, the vesicles are prepared by a method comprising the following steps: taking a cell suspension with a density of 1×10 ⁶ cells/mL, placing an ultrasonic probe in the center of the liquid surface, and performing ultrasonic treatment with an ultrasonic amplitude parameter range of 20%, a time parameter range of 15s, on 2s, and off 2s; then transferring the liquid to a centrifuge tube for centrifugation with centrifugation parameters of 2000g×10min and 20000g×30min, and collecting the supernatant; transferring the supernatant to an ultracentrifuge tube for centrifugation with centrifugation parameters of 150000g×70min.

In some embodiments of the present invention, the product is a cosmetic or medical product for use on the skin or skin appendages.

Specifically, the skin is epidermis, dermis or subcutaneous tissue, especially epidermis.

Specifically, the skin is located on the subject's face (eg, forehead, cheeks, around the eyes), neck, hands, arms, back, legs, abdomen, or any combination thereof.

In particular, the subject is a human.

Specifically, the skin appendages are hairs, sweat glands, sebaceous glands, fingernails or toenails.

Specifically, the aging includes endogenous aging (chronological aging) and exogenous aging, especially photoaging.

In one embodiment of the present invention, the repair is after-sun repair.

Specifically, the anti-aging can be, for example, to reduce the dermatological signs of chronological aging, photoaging, and/or hormonal aging; prevent and/or reduce the appearance of fine lines and/or wrinkles; reduce the noticeability of facial fine lines and wrinkles, facial wrinkles on the cheeks, forehead, vertical wrinkles between the eyes, horizontal wrinkles above the eyes and around the mouth, marionette lines and in particular deep wrinkles or folds; prevent, reduce and/or reduce the appearance and/or depth of fine lines and/or wrinkles; improve the appearance of suborbital and/or periorbital fine lines; reduce the appearance of crow's feet; rejuvenate and/or revitalize the skin, in particular aged skin; reduce skin fragility; prevent and/or reverse the loss of glycosaminoglycans and/or collagen; improve the effects of estrogen imbalance; Prevent skin atrophy; prevent, reduce and/or treat hyperpigmentation; minimize skin hypopigmentation; improve skin tone, radiance, clarity and/or firmness; prevent, reduce and/or improve skin sagging; improve skin firmness, fullness, flexibility and/or softness; improve procollagen and/or collagen production; improve skin texture and/or promote retexturization; improve skin barrier repair and/or function; improve the appearance of skin contours; restore skin radiance and/or brightness; minimize dermatological signs of fatigue and/or tension; combat environmental stressors; replenish components in the skin that are reduced due to aging and/or menopause; improve skin cell-to-cell communication; increase cell proliferation and/or reproduction; increase Reduced skin cell metabolism due to aging and/or menopause; slowing down cell aging; improving skin moisturizing; increasing skin thickness; increasing skin elasticity and/or resilience; enhancing epidermal exfoliation; improving microcirculation; reducing and/or preventing the formation of cellulite; and any combination thereof.

Specifically, the dermatological signs of chronological aging, photoaging, and hormonal aging include, for example, skin sagging, wrinkles, atrophy, red blood streaks, enlarged pores, dull skin color, spots, and the like.

Specifically, the product can be in the form of liquid, solid or semi-solid preparations.

In some embodiments of the present invention, the product may be a preparation suitable for transdermal use, such as a solution, lotion, ointment, paste, patch, gel, film, spray, powder, and the like.

In other embodiments of the present invention, the product may be a preparation suitable for injection, such as a solution, a powder injection, and the like.

Specifically, the product also contains excipients acceptable in the field of cosmetics or medical beauty products, such as one or more of emulsifiers, emollients, humectants, thickeners, solubilizers, surfactants, softeners, preservatives, antioxidants, fragrances, colorants, pH regulators, osmotic pressure regulators, solvents (such as water for injection), buffers, antiallergic agents, anti-inflammatory agents, etc.

In some embodiments of the present invention, the product is a medical beauty product, which is an injection and also contains a sterile solvent such as water for injection or sodium chloride injection, or a sterile buffer such as PBS, Tris-HCl buffer, HEPES buffer, amino acid buffer, etc.

In some embodiments of the present invention, the medical beauty product also contains a second (cosmetic) active ingredient, such as one or more of hyaluronic acid (HA) or its salts, polylactic acid, polycaprolactone, collagen, etc., to achieve a combined effect (such as shrinking pores, brightening skin tone, etc.).

In some embodiments of the present invention, the medical cosmetic product further comprises a local anesthetic, such as lidocaine, bupivacaine, ropivacaine, tetracaine, and the like.

Specifically, the medical cosmetic product can be administered to the subject by injection, topically, or transdermally; the injection can be intradermal injection, intramuscular injection, or subcutaneous injection; the topical or transdermal administration can be accomplished by ion introduction, ultrasound introduction, electroporation, mechanical pressure, osmotic pressure gradient, bandaging therapy, microinjection, high-pressure needle-free injection, microelectronic patch, or any combination thereof.

In some embodiments of the present invention, the product is a cosmetic, for example, a facial cleanser, soap, skin softener, toner, skin care milk, gel, cream, essence, mask, sunscreen, sunscreen spray, gel, liquid foundation, neck cream, body lotion.

In particular, the product can be incorporated into the following containers: capsules, vials, syringes, prefilled syringes, nonwoven fabrics.

Specifically, the product may also contain other active agents, for example, cyclic adenosine monophosphate synthesis stimulators, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, NO synthase inhibitors, 5α-reductase inhibitors, lysyl and/or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and/or anti-air pollution agents, active carbonyl scavengers, anti-glycation agents, antihistamines, antivirals, antiparasitics, α-hydroxy acids, β-hydroxy acids, epidermal hydrolases, vitamins, agents for reducing or treating under-eye bags, exfoliating agents, antimicrobial agents, antifungals, antifungal agents, Bactericides, antibacterial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or are able to prevent their degradation, collagen synthesis stimulators, elastin synthesis stimulators, decorin synthesis stimulators, laminin synthesis stimulators, defensin synthesis stimulators, chaperone synthesis stimulators, aquaporin synthesis stimulators, hyaluronic acid synthesis stimulators, fibronectin synthesis stimulators, deacetylase synthesis stimulators, agents that stimulate the synthesis of lipids and stratum corneum components, agents that stimulate ceramide synthesis, agents that inhibit collagen degradation, agents that inhibit elastin degradation, agents that inhibit serine proteases (such as cathepsin G), agents that stimulate fibroblast proliferation, agents that stimulate keratinogenesis agents that stimulate cell proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that stimulate adipocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that inhibit acetylcholine receptor aggregation, agents that inhibit muscle contraction, glycosaminoglycan synthesis stimulators, anti-hyperkeratosis agents, comedolytic agents, anti-psoriatic drugs, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, agents for the treatment and/or care of sensitive skin, firming agents, anti-stretch mark agents, agents for regulating sebum production, lipolytic agents or agents that stimulate lipolytic agents, lipolytic agents (anti cell ulite agents), antiperspirants, agents stimulating healing, healing agent adjuvants, agents stimulating epidermal cell regeneration, epidermal cell regeneration agent adjuvants, cytokine growth factors, calming agents, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and/or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, venotonic agents, agents acting on cell metabolism, agents improving dermal epidermal junctions, agents inducing hair growth, hair growth inhibitors or blockers, etc., and mixtures thereof, provided that they are physically and chemically compatible with the other ingredients of the composition. Additional examples can be found in the International Cosmetic Ingredient Dictionary & Handbook of the Cosmetic, Toiletries and Fragrance Industry Association (CTFA), 12th Edition (2008).

In a second aspect of the present invention, an anti-aging and photoaging skin repair product is provided, comprising the intracellular nanovesicles described in the first aspect of the present invention.

Specifically, the product is a cosmetic or medical beauty product, which has the definition described in the first aspect of the present invention.

In a third aspect of the present invention, a method for improving the condition or aesthetic appearance of skin is provided, comprising the step of applying the mesenchymal stem cell-derived intracellular nanovesicles described in the first aspect of the present invention or the product described in the second aspect of the present invention to the skin.

Specifically, the administration route may be injection or transdermal administration.

In other embodiments of the invention, the method is a non-disease preventive or therapeutic method (eg, a cosmetic method).

In particular, the improvement in the condition or aesthetic appearance of the skin can be, for example, reducing dermatological signs of chronological, photoaging, hormonal and/or actinic aging; preventing and/or reducing the appearance of fine lines and/or wrinkles; reducing the noticeability of facial fine lines and wrinkles, facial wrinkles on the cheeks, forehead, vertical wrinkles between the eyes, horizontal wrinkles above the eyes and around the mouth, marionette lines and in particular deep wrinkles or folds; Prevent, alleviate and/or reduce the appearance and/or depth of fine lines and/or wrinkles; improve the appearance of suborbital and/or periorbital fine lines; reduce the appearance of crow's feet; rejuvenate and/or revitalize the skin, especially aging skin; reduce skin fragility; prevent and/or reverse the loss of glycosaminoglycans and/or collagen; improve the effects of estrogen imbalance; prevent skin atrophy; prevent, alleviate and/or treat hyperpigmentation; minimize skin hypopigmentation; improve skin tone, radiance, clarity and/or firmness; prevent, alleviate and/or improve skin sagging; improve skin firmness, fullness, flexibility and/or softness; improve the appearance of procollagen and/or collagen Pro-protein production; improving skin texture and/or promoting re-texturization; improving skin barrier repair and/or function; improving the appearance of skin contours; restoring skin radiance and/or brightness; minimizing dermatological signs of fatigue and/or tension; combating environmental stress; replenishing components in the skin that are reduced due to aging and/or menopause; improving skin cell-to-cell communication; increasing cell proliferation and/or reproduction; increasing skin cell metabolism that is reduced due to aging and/or menopause; delaying cell aging; improving skin moisturization; increasing skin thickness; increasing skin elasticity and/or resilience; enhancing epidermal exfoliation; improving microcirculation; reducing and/or preventing the formation of cellulite; and any combination thereof.

Specifically, the dermatological signs of chronological aging include, for example, spots, sagging, wrinkles, red blood streaks, enlarged pores, dull skin tone, and the like.

In a fourth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from adult stem cells (eg, mesenchymal stem cells) in the preparation of a drug for preventing and/or treating diseases caused by photoaging.

Specifically, the intracellular nanovesicles are those described in the first aspect of the present invention.

In one embodiment of the present invention, the application is the use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of drugs for preventing diseases caused by photoaging.

Specifically, the diseases include, but are not limited to, solar dermatitis, photosensitive dermatitis, actinic fibrosis (e.g., rhomboid skin, disseminated fibroelastoma, nodular fibroelastosis, lemon-colored skin, collagenous plaques on the hands and feet, and elastic fibrous nodules on the ears), seborrheic keratosis, actinic granuloma, colloid milia, stellate pseudoscars, hyperpigmentation, melasma, solar lentigo, solar keratosis, skin sun disease, skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, malignant lymphoma, Bowen's disease, eczematoid carcinoma, fibrosarcoma, sweat gland carcinoma, dermatofibrosarcoma protuberans, angiosarcoma, Merkel cell carcinoma), etc.

Specifically, the drug further comprises one or more pharmaceutically acceptable excipients.

Specifically, the pharmaceutically acceptable excipients can be selected from: disintegrants, binders, lubricants, suspending agents, stabilizers, fillers, absorption promoters, surfactants, flavoring agents, antioxidants, preservatives, thickeners, solubilizers, surfactants, preservatives, antioxidants, flavors, colorants, pH regulators, osmotic pressure regulators, solvents (such as water for injection), buffers, antiallergic agents, anti-inflammatory agents, etc. One or more of the following.

Specifically, the drug can be administered by any suitable route, such as enteral administration or parenteral administration (eg, intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, rectal, etc.), especially injection.

Specifically, the drug can be in any suitable dosage form, such as a dosage form for gastrointestinal administration, for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; a dosage form for non-gastrointestinal administration, for example, an injectable dosage form: such as an injection (for example, for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intraperitoneal injection), a dosage form for respiratory administration: such as a spray, an aerosol, a powder aerosol, etc., a dosage form for skin administration, such as an external solution, a lotion, an ointment, a plaster, a paste, a patch, etc., a dosage form for mucosal administration: such as eye drops, eye ointments, nasal drops, gargles, sublingual tablets, etc., a dosage form for cavity administration: such as suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

In some embodiments of the present invention, the drug is an injection, such as a subcutaneous injection or an intradermal injection.

In other embodiments of the present invention, the drug is a transdermal preparation, such as a solution, a lotion, an ointment, a paste, a patch, a gel, a film, and the like.

Specifically, various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field, such as mixing the active ingredient with one or more pharmaceutically acceptable excipients and then preparing the mixture into the desired dosage form.

In the fifth aspect of the present invention, a method for preventing and/or treating diseases caused by photoaging is provided, which comprises the step of administering the mesenchymal stem cell-derived intracellular nanovesicles described in the first aspect of the present invention or the drug described in the fourth aspect to a subject in need thereof.

Specifically, the administration route can be any suitable route, in particular injection, such as subcutaneous injection, intradermal injection.

In particular, the subject is a human.

In a sixth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from adult stem cells (particularly mesenchymal stem cells) in the preparation of a product that promotes hair follicle regeneration.

Specifically, the intracellular nanovesicles are those described in the first aspect of the present invention.

In some embodiments of the present invention, the product is a cosmetic or medical cosmetic product.

Specifically, the product can be in the form of liquid, solid or semi-solid preparations.

Specifically, the product also contains excipients acceptable in the field of cosmetics or medical beauty products, such as one or more of emulsifiers, emollients, humectants, thickeners, solubilizers, surfactants, softeners, preservatives, antioxidants, fragrances, colorants, pH regulators, osmotic pressure regulators, solvents (such as water for injection), buffers, antiallergic agents, anti-inflammatory agents, etc.

In some embodiments of the present invention, the product is a medical cosmetic product, which is an injection or a skin topical preparation such as a gel.

In some embodiments of the present invention, the product is a cosmetic, for example, a hair tonic, a conditioner, a hair lightener, a hair lotion, a hair nutrition Emulsion, shampoo, conditioner, hair treatment cream, hair milk, hair nourishing cream, hair moisturizing cream, hair massage cream, hair wax, hair aerosol, hair mask, hair nourishing film, shampoo soap, shampoo milk, hair dryer, hair preservation treatment agent, hair dye, hair waving agent, hair bleaching cream, hair spray, hair glaze, hair styling agent, hair adhesive, hair moisturizing agent, hair mousse, hair spray.

Specifically, the product can be incorporated into the following containers: vials, syringes, prefilled syringes, and the like.

In a seventh aspect of the present invention, a product for promoting hair follicle regeneration is provided, comprising the intracellular nanovesicles described in the first aspect of the present invention.

Specifically, the product is a cosmetic or medical beauty product, which has the definition described in the fifth aspect of the present invention.

In an eighth aspect of the present invention, a method for promoting hair follicle regeneration is provided, comprising the step of applying the intracellular nanovesicles described in the first aspect of the present invention or the product described in the seventh aspect of the present invention.

Specifically, the administration route may be injection or transdermal administration.

In some embodiments of the invention, the method is a non-disease preventive or therapeutic method (cosmetic method).

In a ninth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from adult stem cells (eg, mesenchymal stem cells) in the preparation of a drug for preventing and/or treating hair loss.

Specifically, the intracellular nanovesicles are those described in the first aspect of the present invention.

Specifically, the hair loss includes senile alopecia, alopecia areata, chemotherapy-induced alopecia, and seborrheic alopecia.

In a tenth aspect of the present invention, a method for preventing and/or treating hair loss is provided, comprising the step of applying the intracellular nanovesicles described in the first aspect of the present invention to the skin.

The present invention provides an application of intracellular nanovesicles (sIVs) derived from adult stem cells (e.g., mesenchymal stem cells) in the anti-aging, repair and regeneration of skin and/or skin appendages. The inventor prepared intracellular nanovesicles (sIVs) derived from mesenchymal stem cells, and found through experiments that sIVs can reduce the increase in ROS levels after UV irradiation, reduce the expression of aging-related molecules (e.g., p21, p53, MMP-1, MMP-3), increase the expression of type I collagen (COL-I) molecules, increase the proliferation activity of human skin fibroblasts, increase SOD activity, effectively improve cell aging (including endogenous aging (natural aging) and exogenous aging (e.g., photoaging)), and can also effectively improve the aging of animal models. It is expected that sIVs can effectively improve skin conditions such as skin sagging, dryness, roughness, deepening of wrinkles, pigmentation, etc. caused by aging, and can also be used to prevent diseases caused by exogenous aging such as solar keratosis, skin cancer, etc. In addition, sIVs can also promote the development and regeneration of hair follicles, help regenerate more hair, and are expected to be used for the prevention and treatment of hair loss. In summary, the sIVs prepared by the present invention have very good application prospects and research value.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG2 shows the optimization process of the separation parameters of intracellular nanovesicles. FIG2A-2B respectively show the protein yield (FIG2A) and vesicle yield (FIG2B) of sIVs obtained at different action times and an ultrasonic amplitude of 20%; at an ultrasonic amplitude of 20%, the action time is less than 10 seconds or greater than 20 seconds, and the vesicle yield drops sharply. FIG2C-2D respectively show the protein yield (FIG2C) and vesicle yield (FIG2D) of sIVs obtained at different ultrasonic amplitudes and an ultrasonic action time of 15s; at an ultrasonic time of 15s, the ultrasonic amplitude is higher than 25%, and the vesicle yield drops sharply. FIG2E shows the transmission electron micrographs of sIVs obtained at different ultrasonic amplitudes and an ultrasonic action time of 15s, and the scale bar is 100nm. FIG2F shows the transmission electron micrographs of sIVs obtained at different action times and an ultrasonic amplitude of 20%, and the scale bar is 100nm.

Figure 3 shows transmission electron microscopy images of sEVs and sIVs of MSCs cells. Wide field scale bar: 200 nm; close-up scale bar: 200 nm.

FIG4 shows the results of nanoparticle size analysis, which shows the particle size distribution of MSCs cells and their sEVs and sIVs.

FIG5 shows the statistical analysis results of the particle sizes of MSCs cells and their sEVs and sIVs (**p<0.01, ***p<0.001 indicates significant differences among the groups).

Figure 6 shows the statistical analysis results of the number of sEVs and sIVs vesicles (A) and total protein production of MSCs cells under equal cell numbers (B) (**p<0.01, **p<0.001 and **p<0.0001 indicate significant differences among the groups).

FIG. 7 shows the results of Coomassie Brilliant Blue staining, which shows the protein distribution of MSCs cells and their sEVs and sIVs.

Figure 8 shows the results of Western blot, which shows the expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD81) of MSCs cells.

Figure 9 shows transmission electron microscopy images of sEVs and sIVs of MSCs at different temperatures. Scale bar: 200 nm.

Figure 10 shows the results of nanoparticle size analysis of sEVs and sIVs of MSCs cells at different temperatures (-80°C, 4°C and 37°C). Figure 10A shows the particle size distribution, and Figure 10B shows the results of particle size statistical analysis (*p<0.05, **p<0.01 indicates significant differences between groups, the left three columns of each figure represent MSC-sEVs, and the right three columns represent MSC-sIVs).

FIG. 11 shows the sIVs-specific proteins in MSCs cells, arranged from high to low abundance, showing the top 50 proteins with the highest expression.

Figure 12 shows the results of super-resolution microscopy and total internal reflection fluorescence structured illumination microscopy. Among them, Figure 12A shows the TIRF-SIM mode (showing the cell membrane), which shows that there are CD63-positive areas on the cell membrane surface (left picture), and almost no TMEM214-positive areas are observed (right picture). Figure 12B shows the wide field-2DSM mode (showing the whole cell), which proves that there are CD63-positive (left picture) and TMEM214-positive (right picture) signals in the whole cell. Figure 12C shows a time-division screenshot of the dynamic observation of living cells, with CD63-labeled intracellular late endosomes, sEVs and cell membranes on the left. The arrow at 0s shows that sEVs have just been released from the cell membrane to the outside of the cell, and gradually move away from the cell membrane from 6min to 14min. The right side is TMEM214, showing that sIVs are diffusely distributed in the cell and are not released outside the cell.

FIG. 13 is a Venn diagram showing the total protein species in MSCs cells, sEVs, and sIVs.

FIG. 14 shows the principal component analysis results of the total proteins identified in MSCs cells, sEVs and sIVs.

FIG. 15 is a heat map showing the presence of differentially expressed proteins between sEVs and sIVs of MSCs cells.

FIG. 16 shows a volcano plot showing the top five significantly differentially expressed proteins between sEVs and sIVs of MSCs cells.

FIG. 17 is a heat map showing the differential expression of exosomal markers between sEVs and sIVs of MSCs cells.

FIG. 18 shows a heat map showing the differential expression of organelle markers between sEVs and sIVs of MSCs cells.

FIG. 19 is a heat map showing the differential expression of Clathrin family proteins between sEVs and sIVs of MSCs cells.

FIG. 20 shows the cellular component enrichment analysis of proteins uniquely expressed by sIVs.

FIG. 21 shows the biological process enrichment analysis of sIVs uniquely expressed proteins.

Figure 22 shows the differences in cytokine levels of IL-1β and IGF2 carried by sEVs and sIVs of MSCs detected by protein spectrum. Among them, Figure 22A shows the differences in cytokine levels of IL-1β carried by sEVs and sIVs of MSCs detected by protein spectrum, and Figure 22B shows the differences in cytokine levels of IGF2 carried by sEVs and sIVs of MSCs detected by protein spectrum (*p < 0.05, ***p < 0.001 indicate significant differences between the groups).

Figure 23 shows the differences in the cytokine levels of IGF-1, EGF, IL-10, IL-6 and TNFα carried by sEVs and sIVs of MSCs detected by ELISA. Among them, Figure 23A shows the differences in the cytokine levels of IGF-1 carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23B shows the differences in the cytokine levels of EGF carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23C shows the differences in the cytokine levels of IL-10 carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23D shows the differences in the cytokine levels of IL-6 carried by sEVs and sIVs of MSCs detected by ELISA, and Figure 23E shows the differences in the cytokine levels of TNFα carried by sEVs and sIVs of MSCs detected by ELISA (**p<0.01, ***p<0.001 indicates significant differences between the groups, ns indicates no statistical difference).

FIG. 24 shows the relative RNA abundance in sEVs and sIVs of MSCs cells (ns indicates no statistical difference).

Figure 25 shows the percentage of small non-coding RNAs read from small RNAs of sEVs and sIVs from MSCs cells. miRNA: micro-RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; tRNA: transfer RNA; rRNA: ribosomal RNA.

FIG. 26 is a Venn diagram showing the types of miRNAs contained in sEVs and sIVs of MSCs cells.

FIG. 27 shows the principal component analysis results of the MSCs cell miRNA dataset.

FIG. 28 is a matchstick chart showing the top 10 highly abundant miRNAs in sEVs and sIVs of MSCs cells.

FIG. 29 is a heat map showing that there are many differentially expressed miRNAs between sEVs and sIVs of MSCs cells.

Figure 30 shows a volcano plot showing the top 5 miRNAs differentially expressed between sEVs and sIVs of MSCs cells. The abscissa represents the expression fold change (log2 difference fold) of miRNA between different samples or comparison combinations, and the ordinate represents the significance level of the expression difference.

Figure 31 shows the enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs derived from MSCs. Figure 31A shows the top 10 entries of biological process (BP), cellular component (CC) and molecular function (MF) in GO analysis. Figure 31B shows the KEGG enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of MSCs.

FIG32 shows the types and proportions of metabolites contained in sEVs and sIVs of MSCs cells.

FIG. 33 shows the results of principal component analysis of lipids contained in MSCs cell sEVs and sIVs.

FIG. 34 is a heat map showing the differential lipid types contained in MSCs cell sEVs and sIVs.

Figure 35 shows a lipidome bar graph of the MSCs cell sIVs group versus the sEVs group. The horizontal axis of the graph represents the relative percentage change in the content of each substance in the group. If the relative percentage change in content is zero, it means that the content of the substance in the two groups is the same; if the relative percentage change in content is a positive number, it means that the content of the substance in the sIVs group is higher; if the relative percentage change in content is a negative number, it means that the content of the substance in the sEVs group is higher. The vertical axis of the lipidome bar graph represents the classification information of lipids.

Figure 36 shows the effects of sIVs and sEVs on the proliferation of photoaged cells. Figure 36A shows the effects of different concentrations of vesicles on cell proliferation at 48 hours, and Figure 36B shows the effects of different durations of action on cell proliferation at a concentration of 10 μg/mL.

Figure 37 shows the SA-β-GAL staining results of each group of cells. SA-β-Gal (Senescence-Associated β-Galactosidase) is a senescence-associated β-galactosidase. Blue indicates SA-β-Gal positive cells.

FIG. 38 shows the effects of sIVs and sEVs on the expression of p21, p53 and MMP-3 in photoaged cells at the mRNA level.

FIG. 39 shows the effects of sIVs and sEVs on the expression of p21, p53 and MMP-3 in naturally senescent cells at the mRNA level.

FIG. 40 shows the effects of sIVs and sEVs on the proliferation capacity of naturally senescent cells.

Figure 41 shows the effects of sIVs and sEVs on photoaged skin. Figure 41A shows the HE staining results of mouse skin tissue in each group, scale bar: 50 μm, and Figure 41B shows the effects of sIVs and sEVs on epidermal thickness and dermal thickness.

FIG. 42 shows the Masson staining results of mouse skin tissues in each group, scale bar: 100 μm.

Figure 43 shows the effects of sIVs and sEVs on apoptosis of photoaged skin cells. Figure 43A shows the TUNEL staining results of the skin tissues of mice in each group, scale bar: 50 μm, and Figure 43B shows the apoptosis signal detection results of the skin tissues of mice in each group.

Figure 44 shows the β-galactosidase staining results of frozen sections of mouse skin tissue in each group, scale bar: 50 μm. Figure 44A shows the representative results of β-galactosidase staining, and Figure 44B shows the statistical results. β-galactosidas means β-galactosidase.

FIG. 45 shows the staining results of frozen sections of mouse skin tissues in each group, scale bar: 50 μm.

FIG. 46 shows the detection results of the SOD, MDA, GSH and IL-6 contents in the skin tissues of mice in each group.

FIG. 47 shows the detection results of the MMP-1 and MMP-3 contents in the skin tissues of mice in each group.

Figure 48 shows the effects of sIVs (ADSCs-sIVs) and sIVs (ADSCs-sEVs) derived from adipose mesenchymal stem cells on reactive oxygen species in photoaged cells. Figure 48A shows the fluorescence detection results of each group of cells, scale: 50 μm, and Figure 48B shows the DHE signal intensity detection results of each group of cells.

FIG. 49 shows the effects of ADSCs-sIVs and ADSCs-sEVs on the proliferation capacity of photoaged cells.

FIG. 50 shows the effects of ADSCs-sIVs and ADSCs-sEVs on the proliferation capacity of natural senescent cells.

FIG. 51 shows the effects of ADSCs-sIVs and ADSCs-sEVs on the anti-aging ability of photoaged cells.

Figure 52 shows the effects of ADSCs-sIVs and ADSCs-sEVs on the antioxidant capacity of photoaged mouse skin tissue. Figure 52A shows the fluorescence detection results of mouse skin tissue in each group, scale: 50 μm, and Figure 52B shows the DHE signal intensity detection results of mouse skin tissue in each group.

FIG. 53 shows the effects of bone marrow mesenchymal stem cell-derived sIVs (BMSCs-sIVs) and sEVs (BMSCs-sEVs) on the proliferation capacity of photoaged cells.

FIG. 54 shows the effects of BMSCs-sIVs and BMSCs-sEVs on the proliferation capacity of natural senescent cells.

FIG. 55 shows the effects of BMSCs-sIVs and BMSCs-sEVs on SOD and GSH in photoaged cells.

FIG. 56 shows the effects of BMSCs-sIVs and BMSCs-sEVs on the levels of MMP-1 and MMP-3 in the skin tissue of photoaged mice.

Figure 57 shows the effect of BMSCs-sIVs and BMSCs-sEVs on the expression of β-galactosidas in the skin tissue of photoaged mice, scale bar: 50 μm. Figure 57A shows the representative results of β-galactosidas staining, and Figure 57B shows the statistical results. β-galactosidas represents β-galactosidase.

FIG. 58 shows the hair growth of mice in each group.

Figure 59 shows the immunofluorescence staining results of frozen sections of mice in each group, wherein Figure 59A shows the β-catenin staining results, scale bar: 50 μm, and Figure 59B shows the Ki67 staining results.

FIG60 shows the results of positive expression of β-catenin and Ki67 in each group of mice.

FIG61 shows the results of β-catenin expression detection in the back skin tissue of each group of mice.

Figure 62 shows the results of hair follicle morphology detection of each group of mice. Figure 62A shows the HE staining results of skin tissue sections of each group of mice, scale bar: 50 μm, and Figure 62B shows the detection results of the number of skin hair follicles, skin thickness and hair bulb diameter of each group of mice.

DETAILED DESCRIPTION

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

In the present invention, skin aging is divided into endogenous aging and exogenous aging according to different influencing factors. Endogenous aging refers to natural aging at the gene level over time. Exogenous aging refers to the accumulation of skin damage caused by environmental factors such as ultraviolet radiation, smoking, toxic chemicals, etc., among which ultraviolet radiation is the most important factor leading to exogenous aging.

In the present invention, the term "anti-aging" refers to the use for preventing, delaying and/or improving aging caused by internal factors (including genetic factors) and external factors (including ultraviolet rays, etc.). For example, "anti-aging" refers to preventing and delaying or improving and alleviating skin changes caused by aging, such as increased skin thickness, damaged skin barrier, reduced skin elasticity, wrinkle formation, increased wrinkle depth or number, dry skin, rough skin, and skin damage caused by ultraviolet rays, etc.

The term "skin appendages" is derived from the epidermis during embryogenesis, including hair, sebaceous glands, sweat glands, nails, etc. Among them, hair consists of three parts: hair shaft, hair root and hair follicle. In some embodiments of the present invention, the skin appendage is hair, especially hair follicle.

The term "mammal" includes rats, mice, guinea pigs, rabbits, dogs, monkeys, chimpanzees, humans, etc., especially humans.

The term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting one or more clinical symptoms of a disease after onset of the disease.

The term "prevent" refers to avoiding, minimizing or making the onset or development of a disease difficult by treating it before it occurs.

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Extraction and culture of mesenchymal stem cells (MSC)

Human umbilical cords were provided by Beijing Beilai Biotechnology Co., Ltd. Umbilical cords obtained from normal pregnancies without complications after cesarean section were immediately placed in saline containing penicillin (100U/mL) and streptomycin (100mg/mL) and then transported to the laboratory within 4 hours. After removing residual blood and blood vessels, the obtained umbilical cords were cut into 1-3mm pieces and digested with 0.1% type II collagenase at 37°C for 1 hour. The suspension was then filtered through a 100-mesh sieve to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco's modified Eagle medium/nutrient mixture F12 complete medium. The culture medium contained 10% fetal bovine serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin. The cells were seeded in a T175 culture flask and cultured in a 5% CO ₂ incubator at 37°C. The culture medium was changed every 3 days. When cell confluence reached 80%, cells were passaged at a subculture ratio of 1:2, and cells from P3 to P5 were used for experiments.

Example 2: Preparation of cell-derived nanovesicles

The process flow of the method for producing intracellular nanovesicles is shown in FIG1 .

1. Cell digestion and counting

When the cells grow to 90% confluence, the cell supernatant is aspirated, the cells are washed twice with PBS, the cells are digested with trypsin, and after digestion, the cells are neutralized and washed three times with PBS. The cells are then counted and the cell number is adjusted to 1×10 ⁶ /mL with PBS.

2. Ultrasonic treatment of cells

Take 2 mL of cell suspension with a density of 1×10 ⁶ cells/mL and add it to the bottom of a 50 mL centrifuge tube. Place the ultrasonic probe in the center of the liquid surface. The ultrasonic amplitude parameter is 20%, the time parameter is 15 s, on 2 s, off 2 s, and the centrifuge tube is placed on ice. Then transfer the liquid to a 2 mL centrifuge tube for centrifugation.

3. Collection of Nanovesicles in Small Cells

The centrifugation parameters were 2000g×10min, 20000g×30min. The supernatant was then collected and transferred to an ultracentrifuge tube, and the centrifugation parameters were 150000g×70min. All the above operations were performed on ice. The obtained precipitate was resuspended in PBS and was small intracellular nanovesicles (sIVs).

4. Collection of small extracellular vesicles

FBS is a necessary condition for culturing cells in vitro, but it contains a large number of bovine extracellular vesicles, which will also be present in complete cell culture medium containing FBS. In order to remove bovine extracellular vesicles, FBS was centrifuged at 110,000×g at 4°C overnight (about 12 hours). When cell fusion reached 60%, the cells were cultured in complete culture medium containing 10% FBS to remove exosomes for 48 hours. Then, the supernatant was collected and extracellular vesicles were separated by ultracentrifugation at 4°C. The specific steps include 300g×10min, 2000g×10min, 10000g×30min and 110000g×70min twice. The obtained precipitate was resuspended in PBS, which was small extracellular vesicles (sEVs) mainly composed of exosomes.

Example 3: Optimization of isolation parameters for intracellular nanovesicles

Referring to the steps of Example 2, the optimization process of the separation parameters of intracellular nanovesicles is shown in FIG2 .

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

Take the sIVs resuspended in PBS, dilute it to 1 ml with PBS, and use the Nanoparticle Tracking Analysis (NTA) software NTA 3.3Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the Mode particle size result. The camera mode is sCMOS. The laser type is Blue488, and the viscosity is 0.9cP.

2. Observation of the morphology of sIVs in different cells by transmission electron microscopy

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

Figures 2A and 2B show the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasonic amplitude of 20%, with an action time of 5s, 10s, 15s, 20s, 25s, 30s and 60s, respectively. The results show that when the action time is less than 10 seconds or more than 20 seconds, the number of vesicles and protein yield obtained drop sharply. Figures 2C and 2D show the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasonic action time of 15s, with an amplitude of 20%, 25%, 30%, 35% and 40%, respectively. The results show that when the ultrasonic amplitude is higher than 25% at an ultrasonic time of 15s, the vesicle yield drops sharply. Figure 2E shows a transmission electron micrograph of sIVs obtained according to the implementation steps at an ultrasonic time of 15s, with an ultrasonic amplitude of 20%, 25%, 30%, 35% and 40%. FIG2F shows transmission electron micrographs of sIVs obtained according to the present implementation steps at an ultrasonic amplitude of 20% and ultrasonic times of 5 s, 10 s, 15 s, 20 s, 25 s, 30 s and 60 s.

This optimization process shows that at an ultrasonic amplitude of 20%, the vesicle yield drops sharply when the action time exceeds 10 seconds or 20 seconds. At an ultrasonic time of 15 seconds, the vesicle yield drops sharply when the ultrasonic amplitude is higher than 25%. 20% amplitude and 15 seconds ultrasonic time are the best parameters for collecting intracellular nanovesicles.

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

1. Experimental instruments and materials

1.1 Experimental Reagents

Table 1 Experimental reagents

1.2 Experimental instruments

Table 2 Experimental instruments

2. Experimental methods

2.1 Transmission electron microscopy observation of the morphology of sIVs and sIVs

Take the sEVs and sIVs (prepared in Example 2) after centrifugation, resuspend them in 200 μL PBS solution and mix them evenly, take 10 μL of sEVs and sIVs solution and mix them with 4% PFA in a volume ratio of 1:1, drop them on a clean plastic film to form droplets, then put the front side of the carbon mesh on the droplets and leave it for 20 minutes, negatively stain with 10 μL phosphotungstic acid for 90 seconds, bake the carbon mesh dry, and observe it using a HitacW-7500 transmission electron microscope.

2.2 Nanoparticle size analysis to detect the particle diameter of sEVs and sIVs

Take the sEVs and sIVs resuspended in PBS, dilute them to 1 ml with PBS, and use Nanoparticle Tracking Analysis (NTA) 3.3Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue 488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the particle size result of Mode. The camera mode is set to sCMOS. The laser type is set to Blue488, and the viscosity is set to 0.9cP.

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

After BCA quantification, take an equal amount of protein sample, add PBS to 20μl, add 5μl protein loading buffer (5×), heat at 95℃ for 5 minutes. The protein electrophoresis conditions are 100V, 90min. After electrophoresis, add Coomassie Brilliant Blue Ultrafast Staining Solution, incubate at room temperature for 2 hours, and wash with pure water until the water becomes clear. Take a picture of the gel.

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

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

3. Statistical processing

The experimental data are expressed as mean ± standard deviation. Indicates. All experimental data were tested for normality. SPSS22.0 was used to analyze all quantitative data. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, non-parametric tests were used, and P values < 0.05 were considered statistically significant.

4. Experimental results

4.1 Transmission electron microscopy reveals the morphology of sEVs and sIVs

MSCs cells were enriched for sIVs and detected using transmission electron microscopy. As shown in Figure 3, MSCs cells' sEVs were round or horseshoe-shaped with a diameter of 100-200 nm, and sIVs were numerous, round in shape, and less than 100 nm in diameter. Electron microscopy results showed that the diameter of sIVs was significantly smaller than that of sEVs.

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

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

After statistical analysis, the results are shown in Figure 5. The average particle size of sEVs of MSC is 123.1±4.453nm, and the average particle size of sIVs is 75.28±9.067nm; the particle size of sIVs is smaller than that of sEVs.

4.3 Comparison of the yields of sEVs and sIVs at equal cell weights

To compare the yield of the two types of vesicles, we collected sEVs and sIVs from cell culture supernatants and adherent cells at the same time. After NTA detection, the results showed that the number of sIVs vesicles produced by 1×10 ⁷ cells was 10 to 20 times higher than that of sEVs (Figure 6A), and the protein products in sIVs derived from 1×10 ⁷ cells were 20 to 40 times higher than that of sEVs (Figure 6B). This shows that the yield of sIVs is much higher than that of sEVs.

4.4 Differences in protein distribution between sEVs and sIVs

The whole proteins of cells, sEVs and sIVs were separated by SDS-PAGE, and the total protein distribution was displayed by Coomassie Brilliant Blue staining. The staining results showed that the proteins contained in cells showed the most abundant bands, with multiple high-abundance protein bands; sEVs contained fewer types of proteins, and the high-abundance proteins were located around 200kD and 70kD; sIVs contained more types of proteins than sEVs, and the high-abundance proteins were located around 250kD and 55kD (Figure 7). The protein distribution between different cells was different, which preliminarily indicated that sEVs and sIVs had different protein components and were different from the total protein distribution of cells and sEVs.

To further analyze the protein expression characteristics of cells and their sEVs and sIVs. Western blot was used to detect the expression of exosome marker proteins, Alix, HSP70, TSG101, CD63, and CD81 of cells and their sEVs and sIVs under equal protein conditions. The results are shown in Figure 8. The sEVs of cells express the most exosome marker proteins, and the cells themselves also express a certain amount of Alix, HSP70, TSG101, and CD63; however, Alix and CD81 of sIVs are basically not expressed, and the expression levels of HSP70, TSG101 and CD63 are also much lower than those of cells and sEVs, which further shows that sIVs do not have the characteristics of exosomes and are not the precursors of exosomes in cells.

4.5 Comparison of the stability of sEVs and sIVs at different temperatures

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

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

To gain a deeper understanding of the intracellular distribution of sIVs, we used proteomic analysis to identify proteins uniquely expressed in sIVs compared to sEVs, namely IV characteristic proteins, so that the characteristic proteins carry green fluorescent protein and perform intracellular imaging to visualize the morphology of sIVs in cells. By performing proteomic analysis on sIVs and sEVs, proteins uniquely expressed in sIVs were identified. The protein expression abundance was sorted from high to low, and the top 50 proteins were displayed (Figure 11). We observed that the expression abundance of TMEM214 protein was the highest in MSCs. TMEM214 is a transmembrane protein involved in vesicle trafficking and protein trafficking (Zhao J., Xu J., Wang Y., et al. Membrane Localized GbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt. Plants (Basel). 2022 Sep 8; 11(18): 2342.). Therefore, we used green fluorescent protein GFP to mark TMEM214 to visually display the status of sIVs in cells, and used GFP-labeled CD63 as a marker for sEVs in cells.

Super-resolution microscopy combined with total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) showed that CD63 was visible on the cell membrane (Figure 12A, green), and TMEM214 was not expressed on the cell membrane (Figure 12A, red). This finding excludes the possibility that sIVs originate from cell membrane reconstitution and confirms that the origin and activity site of sIVs are located inside the cell. Super-resolution microscopy using Wildfield-2DSM scanning mode provides an overview of protein expression throughout the cell, and images show that both CD63 and TMEM214 are significantly expressed in the cell (Figure 12B). Subsequently, under wide-field conditions, we observed the dynamic changes of these protein-labeled structures in living cells. Pictures were taken every 10 seconds for 15 minutes to form a dynamic video. In the video screenshot, sEVs can be observed from the cell membrane. The process of dynamic release to the extracellular space (Figure 12C, green, white arrow), while TMEM214-labeled sIVs were diffusely distributed in the cells and were not released outside the cells (Figure 12C, red). Intuitive microscopic imaging showed that sIVs were diffusely distributed in the cells in a cloud-like manner and were not released outside the cells.

5. Summary

In this example, we took MSCs cells as an example to collect and characterize sIVs and sEVs. Through transmission electron microscopy and nanoparticle size analysis, it was found that the size of sIVs was significantly smaller than that of sEVs; the total protein expression patterns of cells, sEVs and sIVs were different, and sIVs expressed low exosome marker proteins; under the same number of cells, the yield of sIVs was significantly more than that of sEVs. Under -80°C conditions, the stability of sIVs and sEVs was comparable, but under 37°C conditions, the stability of sIVs was significantly better than that of sEVs. Super-resolution imaging was used to observe that sEVs were released to the outside of the cell through the cell membrane, while sIVs were frequently active inside the cell. In general, the sIVs vesicles collected by the method described in the present invention contain a unique protein composition, are highly stable at physiological temperature, and have a yield much higher than that of extracellular vesicles.

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

1. Experimental instruments and materials

1.1 Experimental Reagents

Table 3 Experimental reagents

1.2 Experimental instruments

Table 4 Experimental instruments

2. Experimental methods

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

2.1.1 Protein extraction

1) 220ul of urea lysis solution (8M urea, _{50mM NH4HCO3} , protease inhibitor) was added to cells, sEVs and sIVs (prepared in Example 2) respectively, and lysed at room temperature for 5 minutes.

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

3) The protein concentration was detected by BCA method, and 100 μg protein was taken from each of cells, sEVs, and sIVs.

2.1.2 Protein denaturation

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

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

3) First, mark the sample number on the collection tube, and balance the 10kDa ultrafiltration tube twice with HPLC grade methanol, each time with a volume of 150μl of methanol. 14,000g for 5 min, then add 300μl 50mM NH ₄ HCO ₃ , then rinse twice, add 100μg of the reduced alkylated protein sample, centrifuge at 14,000g for 20 min at 4℃, add 300μl 50mM NH ₄ HCO ₃ and rinse three times, replace a new collection tube, and add 75μl 50mM NH ₄ HCO ₃ to the ultrafiltration tube.

2.1.3 Protease cleavage

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

2) The next day, centrifuge at 14,000 g for 20 minutes at 4°C, add 50 μl of 50 mM NH ₄ HCO ₃ , rinse twice, add 1% (volume ratio) formic acid to the collection tube to terminate the enzyme cleavage, and evaporate to dryness at 60°C in a vacuum.

2.1.4 Library construction and fractionation

1) 30 μl of 0.1% formic acid aqueous solution was used to resuspend the sample and the concentration was measured using a nanodrop. Next, approximately 10 μg of peptides were taken from each sample and combined into one sample S.

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

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

Table 5 Fractionation reagent ratio

4) Use wire to load a layer of C18 membrane into a 200 μl pipette tip. Next, dissolve 30 mg of high pH resistant C18 filler in 200 μl of Buffer A and add the filler to the fractionation column. Then, centrifuge at 3,000 g for 2 minutes at 4°C, add 200 μl of Buffer A to wash once, and then add 200 μl of Buffer B to wash three times. Finally, set the column aside for later use.

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

6) Wash the fractionation column 3 times with 200 μl of Buffer B. Then, add 150 μl of elution buffer of different concentrations and perform gradient elution. Combine the 6% and 35% fractions into one fraction, and take 1.5 μg of peptides from each fraction for mass spectrometry analysis in DDA mode.

2.2 HPLC-MS parameters of cells and their sEVs and sIVs

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

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

When performing DDA scanning, the mass spectrometry parameters were set as follows: the accumulation time of TOF MS was 0.25 seconds, the mass scanning range covered 300-1500 Daltons (Da), only ions with valences of +2 to +5 were detected, and the mass deviation was required to be less than 50ppm. A maximum of 60 ions were monitored in each cycle, and after each detection, the detected ions were isolated for 16 seconds. The fragmentation energy mode used the dynamic fragmentation mode. The accumulation time of Product ion was 0.04 seconds, and the high-sensitivity scanning mode was used.

When performing DIA scanning, the mass spectrometry parameters are different: the accumulation time of TOF MS is 0.05 seconds, and the secondary scan uses high sensitivity mode. The number of variable windows is set to 100, the accumulation time of each window is 30 milliseconds, and the mass scan range is also 300-1500Da. The specific mass range of each variable window is calculated using the SWATH Variable Window Calculator_V1.1 program.

2.3 Cell and sEVs, sIVs protein spectrum data processing and bioinformatics analysis

The raw data collected in DDA mode were searched using Proteinpilot software (version 5.0.1) with trypsin as the restriction enzyme. The database used was the Uniprot database, which contains 20,431 annotated proteins and was published in July 2019. The screening criteria were unused protScore greater than 0.05. The search results of Proteinpilot were imported into SWATH software (version 2.0) as a database to quantify the data collected in DIA mode.

During the quantitative process, 6 peptides were selected for each protein, and 6 transitions (ion pairs) were selected for each peptide. The confidence of the peptide was set to 99%, and the FDR (false positive rate) was set to 1%. At the same time, modified peptides were excluded, the peak extraction window was set to 10 minutes, and the mass deviation was controlled within 50ppm. Two endogenous peptides were selected every 10 minutes to correct the retention time, and the output peak area was used as the quantitative value.

The processing of protein expression data involves the following steps: first, the raw quantitative values are log2 transformed to satisfy the normal distribution, and then The normalization was performed using the normalize.quantiles function in the preprocessCore package of the R language. After removing proteins without gene names, the R language stats package was used for differential analysis, and proteins with a P value less than 0.05 and a change fold greater than 1.5 were selected as differential proteins. At the same time, the corrected p value was set to less than 0.05.

GO and pathway analysis were performed using the Cytoscape plug-in clueGo. In GO enrichment analysis, cell component (CC), molecular function (MF), and biological process (BP) were selected for analysis. In pathway enrichment analysis, the kegg and reactome databases were selected for analysis.

Heatmap, principal component analysis (PCA) score, Venn diagram, volcano map, etc. were performed using R language or drawn using Hiplot software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Metascape online analysis software.

2.4 ELISA

Collect sEVs and sIVs of MSCs, adjust to equal mass, and operate according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit. Add the sample to the wells coated with antibodies, incubate for 2 hours, and wash the wells; add biotin, incubate for 1 hour, wash the wells; add HRP, incubate for 1 hour, wash the wells; add TMB substrate, incubate for 20 minutes; add STOP solution until the color is obvious. Use an enzyme reader to read the absorbance value of each well in the well plate, using a wavelength of 450nm/540nm.

3. Statistical processing

The experimental data are expressed as mean ± standard deviation. The experimental data were tested for normality. SPSS 22.0 was used to analyze all quantitative data, and t test was used for statistical analysis between the two groups. P value < 0.05 was considered statistically significant.

4. Experimental results

4.1 Venn diagram showing differences in protein composition of cells and their sEVs and sIVs

In order to characterize the molecular composition of sEVs and sIVs, we used label-free mass spectrometry to perform proteomic analysis of sEVs and sIVs derived from MSCs cells and compared them with cells. In MSCs cells, 2744 proteins were identified; at the same time, 1678 proteins were detected in sEVs and 2066 proteins were found in sIVs (Figure 13). There is some overlap in the types of proteins between them, but they are not exactly the same. The protein content in sIVs is more diverse than that in sEVs.

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

PCA analysis of the protein components of cells and their sEVs and sIVs (Figure 14) showed that cells, sEVs and sIVs showed different protein distribution patterns. sEVs and sIVs showed significant differences in protein expression. This indicates that sIVs are different from sEVs and have unique protein expression characteristics.

4.3 Quantifiable protein differential analysis of cells and their sEVs and sIVs

Further statistics showed that there were 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, of which 753 were significantly downregulated in sIVs compared with sEVs, 672 were significantly upregulated, 468 of which had a difference of more than 10 times, 227 were significantly downregulated in sIVs compared with sEVs, and 241 were significantly upregulated (Figure 15), further demonstrating the uniqueness of sIVs.

The most significantly upregulated and downregulated proteins in sEVs and sIVs were not exactly the same (Figure 16). However, membrane-associated proteins were expressed at lower levels in sIVs, while endoplasmic reticulum and ribosome-associated proteins were more abundant in sIVs. This initially suggests that sIVs are unique entities produced by cells, rather than precursors or fragments of cell lysates or extracellular vesicles.

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

We extracted the list of exosome markers recommended by MISEV2018 (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.) and compared sEVs and sIVs with these markers. sEVs showed higher expression levels of exosome markers, while sIVs had lower expression levels of most exosome marker proteins (Figure 17). This further suggests that sIVs do not have exosome characteristics and are unique vesicles from the inside of cells.

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

We also performed a comparative analysis of the organelle protein expression profiles of sEVs and sIVs. Overall, sIVs contained higher levels of intracellular organelle proteins than sEVs (Figure 18). In particular, sIVs showed increased expression levels of proteins associated with membrane-enriched organelles such as endosomes, endoplasmic reticulum, and Golgi apparatus. In contrast, sEVs contained more abundant cellular membrane proteins (Figure 18). This suggests that sIVs have intracellular characteristics.

4.6 Differential expression of Clathrin protein family in cells and their sEVs and sIVs

Among the proteins contained in intracellular vesicles, the Clathrin protein family is essential for the organization and activity of vesicles. Clathrin proteins play a key role in intracellular transport by promoting cargo transport between organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways. Given the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that most clathrin family proteins were upregulated in sIVs, while sEVs showed low expression (Figure 19). This finding suggests that the sIVs we isolated may participate in the communication between different cellular compartments inside the cell.

4.7 Enrichment analysis of sIVs-specific proteins

In Example 4, we analyzed and obtained 106 proteins that are different from those expressed by sIVs and sEVs. These proteins are unique proteins expressed by sIVs and can represent the characteristics of sIVs. We performed gene enrichment analysis on these proteins. In terms of cellular components (CC), these proteins are related to COPII-coated endoplasmic reticulum to Golgi transport vesicles, transport vesicles, coated vesicles, endoplasmic reticulum to Golgi transport vesicle membranes, endoplasmic reticulum to Golgi intermediate compartments, transport vesicle membranes, coated vesicle membranes, etc. (Figure 20). At the same time, 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. (Figure 21). This one The results showed that the sIVs we isolated were intrinsic vesicle components within cells.

Comparison of cytokines contained in sEVs and sIVs

Intracellular vesicles are involved in the intracellular transport of various secretory factors, while exosomes carry these factors to the extracellular space. Therefore, we compared the levels of cytokines carried by sEVs and sIVs. Proteomic analysis showed that sIVs had lower levels of interleukin-1β (IL-1β) and insulin-like growth factor 2 (IGF-2) compared with sEVs (Figure 22). Subsequently, we used ELISA to further quantify low-abundance cytokines. The results showed that sIVs contained high levels of insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and interleukin-10 (IL-10) on the basis of the same mass (Figure 23A-C). At the same time, there was no significant difference in IL-6 levels between sEVs and sIVs (Figure 23D), and the level of Tumor Necrosis Factor α (TNFα) (Figure 23E) in sIVs was lower.

5. Summary

In this example, we used MSCs cells as an example and used proteomics technology to characterize the protein composition of cells, sEVs and sIVs. The results showed that sIVs had a unique protein expression profile, which was different from cells and sEVs; sIVs expressed low exosome markers and highly expressed marker proteins and Clathrin protein family of intracellular membrane-rich organelles, which indicated that sIVs played a role in intracellular material transport and mediated intracellular organelle communication; gene enrichment analysis of sIVs directly suggested that sIVs were involved in the transport of substances between the endoplasmic reticulum and the Golgi apparatus, including forward transport mediated by endoplasmic reticulum to Golgi apparatus vesicles and retrograde vesicle-mediated transport from the Golgi apparatus back to the endoplasm. These findings strongly suggest that sIVs play a key role in the intracellular material transport process, especially mediating the exchange of substances between organelles. Among them, COP-coated vesicles have been widely reported to participate in the intracellular material transport process initiated by the endoplasmic reticulum. During this process, 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 shed their coating and fuse with each other to form tubular clusters of vesicles. The Golgi apparatus is responsible for modifying these proteins and lipids received from the endoplasmic reticulum and distributing them to the cell membrane, endosomes, and secretory vesicles. Proteins and lipids move in the cis-to-trans direction within the Golgi apparatus and complete this process through vesicular transport. Proteomic analysis further confirmed that sIVs is involved in the intracellular vesicular transport process and is closely related to the endoplasmic reticulum, Golgi apparatus, and COP-coated vesicles.

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

Example 6: sIVs have unique miRNA expression profiles

1. Experimental instruments and materials

1.1 Experimental Reagents

Table 6 Experimental reagents

1.2 Experimental instruments

Table 7 Experimental instruments

2. Experimental methods

2.1 Data collection and analysis

2.1.1 RNA isolation, library preparation and sequencing

After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS and tested for RNA degradation and contamination on a 1% agarose gel. Check RNA purity by spectrophotometer. RNA concentration was determined using Measurements were performed using the Qubit ^™ RNA Assay Kit in a Flurometer. Detection was performed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 System.

2.1.2 Library preparation for small RNA sequencing

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

2.1.3 Cluster generation and sequencing

The index was performed using the TruSeq SR Cluster Kit v3-cBot-HS (Illumia) on the cBot Cluster Generation System according to the manufacturer’s instructions. The encoded samples were clustered and sequenced on an Illumina HiSeq 2500/2000 platform to generate 50 bp single-end reads for library preparation.

2.1.4 Data Analysis

1) Quality Control

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

2) Alignment of known miRNAs

The mapped small RNA tags were used to search for known miRNAs. Using miRBase20.0 as a reference, the modified software mirdeep2 ( MR, Mackowiak SD, Li N., et al. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades [J]. Nucleic acids research, 2012, 40 (1): 37-52) and srna-tools-cli were used to obtain potential miRNAs and draw secondary structures. Custom scripts were used to obtain the miRNA counts and base biases at the first position of identified miRNAs with a specific length, as well as the miRNA counts and base biases at each position of all identified miRNAs.

3) Summary of small RNA annotation

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

4) Data analysis

Target gene prediction was performed using miRanda, and miRNA target gene prediction was the intersection of miRanda and RNAhybrid. The differentially expressed miRNA input data was the readcount data obtained from the miRNA expression level analysis. For samples with biological repeatability: the DESeq R package (3.0.3) was used to perform differential expression analysis on the two conditions/groups. The P value was adjusted using the Benjamini&Hochberg method. By default, the corrected P value was set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, Venn diagrams, volcano maps, etc. were drawn online using Hiplot and adjusted using Adobe Illustrator.

5) GO and KEGG enrichment analysis

GO enrichment analysis was performed on the target gene candidates of differentially expressed miRNAs (hereinafter referred to as "target gene candidates"). GO enrichment analysis used GOseq based on Wallenius non-central hypergeometric distribution, which can adjust gene length bias. KOBAS software was used to test the statistical enrichment of target gene candidates in KEGG pathways.

3. Experimental results

3.1 Relative RNA abundance in sEVs and sIVs

First, we analyzed the RNA abundance of sEVs and sIVs using a bioanalyzer ( Figure 24 ).

3.2 Distribution of small RNAs in sEVs and sIVs

Small RNAs related to cells and extracellular vesicles have been a hot topic in recent years, especially microRNAs (miRNAs), which have multiple biological functions and can be used as biomarkers for multiple diseases. We analyzed the types of small RNAs in sEVs and sIVs. The comparison and annotation of small RNAs of various types with total RNA were summarized. Since there is a situation where one sRNA is simultaneously matched with several different annotation information, in order to make each unique sRNA have a unique annotation, small RNAs were classified according to the priority order of known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>novel miRNA detection, and the proportion of each small RNA in total RNA was calculated. The results showed that in sEVs, YRNA is the most important RNA (YRNA is a class of highly conserved small non-coding RNA (see Xie Yuxin, Chen Tianxing, Wang Li, et al. YRNA: Research Progress in Cancer and Non-Cancer [J]. Chinese Journal of Experimental Surgery, 2021, 38(9): 1844-1848.)); in sIVs, miRNA is the most important RNA. MiRNA accounted for 29.15% in sEVs of MSCs and 92.52% in sIVs. sIVs have more abundant small RNA species, among which the content of miRNA is significantly higher than that of sEVs (Figure 25).

3.3 Total miRNA expression characteristics of sEVs and sIVs

MiRNA has a rich biological regulatory role and accounts for a large proportion of small RNA. Therefore, we conducted a follow-up analysis of miRNA and used a Venn diagram to analyze the types of miRNA contained in sEVs and sIVs. The results showed that 694 miRNAs were detected in sEVs and 989 miRNAs were found in sIVs (Figure 26). There is some overlap in the types of miRNAs between them, but they are not exactly the same.

Because sEVs and sIVs contain commonly expressed miRNAs, principal component analysis was subsequently used to compare the miRNA expression patterns of sEVs and sIVs. The results showed that the miRNA components contained in the two vesicles were quite different and the expression patterns were unrelated ( Figure 27 ), further indicating that sIVs are different from sEVs and contain a unique miRNA expression profile.

3.4 Highly abundant miRNAs in sEVs and sIVs

By analyzing the top 10 high-abundance miRNAs in sEVs and sIVs, we found that miR-148a-3p and let-7i-5p were expressed at higher levels in sEVs, and let-7f-5p was expressed at higher levels in sIVs. At the same time, miRNAs such as miR-148-3p, miR-21-5p and miR-100-5p were expressed at higher levels in both sEVs and sIVs (Figure 28).

3.5 Analysis of differentially expressed miRNAs in sEVs and sIVs

In order to further compare the differences in miRNAs between sEVs and sIVs, we performed differential expression analysis on sEVs and sIVs. The results showed that in MSCs, there were 70 differential miRNAs between sEVs and sIVs, of which 22 were significantly downregulated in sIVs compared with sEVs, and 48 were significantly upregulated (Figures 29-30). This further illustrates that sIVs are different from sEVs.

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

The way miRNA exerts its biological effects is to regulate downstream target genes. Therefore, after comparing the differential miRNAs in each group, we performed gene enrichment analysis on the target gene sets of these miRNAs, including GO analysis and KEGG analysis. For the convenience of expression, we will refer to "target genes of differentially expressed miRNAs" as "candidate target genes". The results of GO enrichment analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to intracellular metabolic processes, cell localization was intracellular membrane-related organelles, and molecular functions were related to protein binding and enzyme metabolic reactions (Figure 31A); KEGG pathway analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to pathways such as axon guidance, cell differentiation, endocytosis and immune regulation (T cell receptor signaling pathway, B cell receptor signaling pathway) (Figure 31B).

4. Summary

In this example, we used MSCs cells as the object, and used small RNA sequencing technology to conduct a detailed analysis of the small RNA composition of sEVs and sIVs, and deeply explored the expression pattern of miRNA. After being produced in the nucleus, intracellular miRNA is transported to the cytoplasm and participates in the regulation of target genes. Therefore, miRNA produced in cells must first perform its biological functions by regulating gene expression and participating in various cell biological processes, such as cell proliferation, differentiation and apoptosis. Many miRNAs have been 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, thereby affecting cell survival and apoptosis. They can maintain cell homeostasis by regulating apoptosis-related genes, such as BCL2 family members, caspase and p53. In cells, miRNAs can act as signaling pathway regulators to adjust cell biological processes, such as growth factor signals, responses to oxidative stress and inflammatory responses. They can target key molecules in specific signaling pathways, thereby affecting the entire signal transduction pathway. Our experimental results show that sIVs are rich in miRNAs, which suggests that sIVs may play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis and signaling pathways, and have great application potential. The results showed that sIVs showed a unique miRNA expression profile, which was significantly different from sEVs, and the miRNA content in sIVs was richer. This finding suggests that sIVs have potential biological regulatory effects and may promote information exchange between different organelles in cells. By performing candidate gene enrichment analysis on differentially expressed miRNAs in sEVs and sIVs, we further found that sIVs are closely associated with intracellular membrane-like organelles. In summary, sEVs and sIVs differ in small RNA components, especially miRNAs. Enrichment analysis of miRNAs further confirmed that sIVs play an important role in intracellular material transport. Compared with sEVs, sIVs contain more diverse miRNAs and may have richer biological functions. These findings provide important clues for a deeper understanding of the function of sIVs in cell biology.

Example 7: sIVs have unique lipidomic characteristics

1. Experimental instruments and materials

1.1 Experimental Reagents

Table 8 Experimental reagents

1.2 Experimental instruments

Table 9 Experimental instruments

2. Experimental methods

2.1 Metabolite extraction

After separation and enrichment of sEVs and sIVs (prepared in Example 2), resuspend them in PBS. Add 200 μL of water to the container, followed by 480 μL of an extract solution, which is a mixture of MTBE and MeOH in a ratio of 5:1 and contains an internal standard substance. Quickly place the mixture in a liquid nitrogen tank and freeze for 1 minute. Then take out and thaw, and mix with a vortex mixer for 30 seconds to make the solution uniform. Repeat the above freezing, thawing and mixing steps 3 times, followed by ultrasonic treatment in an ice water bath for 10 minutes. The treated sample was allowed to stand at -40°C for 1 hour. Then, centrifuged at 3000rpm (centrifugal force 900×g, radius 8.6cm) for 15 minutes at 4°C to separate the samples. Take out 300μL from the supernatant, transfer to an EP tube, and vacuum dry. Add 100μL of the complex solution (DCM:MeOH=1:1) to the dried sample, vortex mix for 30 seconds, and ultrasonically treat again in an ice water bath for 10 minutes. Finally, centrifuge at 13000rpm (centrifugal force 16200×g, radius 8.6cm) for 15 minutes at 4°C, take 75μL of the supernatant and transfer it to the injection bottle, ready for machine detection.

2.2 Metabolite detection

Vanquish ultra-high performance liquid chromatograph was used, and Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) liquid chromatography column was used for chromatographic separation of the target compounds. For liquid chromatography, phase A was a 40% aqueous solution and 60% acetonitrile solution containing 10 mmol/L ammonium formate; phase B was a 10% acetonitrile and 90% isopropanol solution containing 50 mL/1000 mL (10 mmol/L) ammonium formate aqueous solution. We used the following gradient elution program: 0-1.0 min, 40% B; 1.0-12.0 min, linear increase to 100% B; 12.0-13.5 min, maintain 100% B; 13.5-13.7 min, linear decrease to 40% B; 13.7-18.0 min, maintain 40% B. The mobile phase flow rate was set at 0.3 mL/min, the column temperature was 55 °C, the sample tray temperature was 4 °C, and the injection volume was 2 μL (positive and negative ion modes).

At the same time, the primary and secondary mass spectrometry data were collected using a Thermo Q Exactive HFX mass spectrometer under the control of Xcalibur control software (version: 4.0.27, Thermo). The specific parameters are as follows: Sheath gas flow rate is set to 10Arb, Capillary temperature is set to 350℃, Full ms resolution is set to 120000, MS/MS resolution is set to 7500, Collision energy is set to 10/30/60 in NCE mode, and Spray Voltage is set to 4kV (positive ion mode) or -3.8kV (negative ion mode).

2.3 Data Analysis

ProteoWizard software was used to convert the mass spectra into mzXML format. XCMS was then used for retention time correction, peak identification, peak extraction, peak integration, and peak alignment, with minfrac set to 0.5 and cutoff set to 0.3. Lipid identification was performed using XCMS software, a self-written R package, and the lipidblast database. Bioinformatics graphics were drawn online using Hiplot and adjusted using Adobe Illustrator.

3. Experimental results

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

3.1 Proportion of various metabolites in sEVs and sIVs

The secondary spectra of lipidomics are accidental, so only lipids identified in all groups in a set of comparative information are credible. Therefore, we classified and counted the metabolites identified by different cells according to the chemical classification information. The proportion of each type of metabolite is shown in Figure 32. MSCs identified 31 types. There are differences in the expression levels of various lipids in sEVs and sIVs. PC and PE are common lipid components of cell membranes. The results in Figure 32 show that the proportion of PE and PC in vesicles is high, which indicates that sEVs and sIVs have a large number of biological membrane structures.

3.2 Total lipid expression characteristics in sEVs and sIVs

Metabolomics data has the characteristics of high throughput, and principal component analysis can effectively highlight the overall distribution trend of metabolomics data and the degree of difference between samples in different groups. The results showed that MSCs cell sEVs and sIVs had different lipid distribution patterns (Figure 33), that is, sIVs is a unique vesicle population, different from sEVs, with significantly different lipid expression patterns.

3.3 Differential lipid expression characteristics in sEVs and sIVs

Heat maps can intuitively display the overall distribution of metabolite differences between groups. We visualized the results of screening differential metabolites in the form of heat maps. The results of the sIVs group versus the sEVs group are shown in Figure 34.

3.4 Changes in the content and classification of differential lipids in sEVs and sIVs

The lipidome bar graph uses the content change degree and classification information of metabolites for visualization. The results of the sIVs group versus the sEVs group are shown in Figure 35. Each column in the lipidome bar graph represents a metabolite. In MSCs, PC, PI, PE, PG, and OxPI are significantly overexpressed in sIVs, among which PC is overexpressed 200 times in sIVs.

4. Summary

Lipidomics identifies and quantifies various lipid molecules. Lipids are divided into eight categories, including fatty acyl, glycerolipids, phospholipids, sterol lipids, propenol lipids, sphingolipids, glycolipids and polyketides. The cell membrane mainly contains various phospholipids, which can be further divided into glycerophospholipids and sphingomyelin, which have obvious differences. Glycerophospholipids are mainly located in the inner leaflet of the phospholipid bilayer in the cell membrane, and together with cholesterol, they constitute the main components of the cell membrane. In this example, sIVs contain more glycerophospholipids, such as PC and PE. Sphingomyelin is a type of phospholipid containing a sphingosine group. Sphingomyelin is located in the outer leaflet of the cell membrane and is mainly involved in neuronal activity and signal transduction. In this example, sEVs contain more sphingomyelin, such as SM. In addition, glycerophospholipids are also involved in many other physiological processes in the body, such as energy metabolism, hormone synthesis, etc.; while sphingomyelin plays a relatively small role in these processes. PC and PE expression levels are high in sIVs, among which PC is more than 200 times higher in sIVs. Among them, PC is also known as lecithin, which is known as the "third nutrient" alongside proteins and vitamins, and plays many important roles in biology. Lecithin can increase the axonal growth of neurons, promote brain development, enhance memory, and prevent Alzheimer's disease. In addition, PE is one of the main molecules that constitute the skeleton of biological membranes. Its unique structure, including a phosphate group, a glycerol, an acyl group, and an ethanolamine, enables it to form stable non-lamellar and multi-layer liposome vesicles in biological membranes. This structure provides a stable foundation for biological membranes and helps maintain the normal structure and function of cells.

Interestingly, PC and PE, which are highly expressed in sIVs, are both glycerophospholipids. The endoplasmic reticulum is the site of glycerophospholipid synthesis, so sIVs contain more glycerol. Oil phospholipids are reasonable, further confirming that sIVs are intracellular components that mediate intracellular material transport and communication between organelles. However, sEVs contain more sphingomyelin. sEVs originate from the invagination of the cell membrane and are secreted to the outside of the cell through the cell membrane, so they contain more components on the outside of the cell membrane. This also shows that sIVs lack an external membrane structure, and the difference in multiple lipids distinguishes sIVs from sEVs. Previous studies have shown that the transport of proteins and lipids in cells is related to membrane curvature and lipid distribution effects. Glycerol phospholipids can regulate the curvature and fluidity of the membrane by adjusting the chain length and cooperating with cholesterol, giving sIVs more vitality, thereby continuously participating in membrane fusion and fission events in cells.

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

The sIVs and sEVs described in the following Examples 8-10 and 13 all use the small intracellular nanovesicles and small extracellular nanovesicles prepared in Example 2.

Example 8: Anti-aging study of sIVs

1. Experimental methods

1.1 Cultivation of human skin fibroblasts (HFF-1)

HFF-1 (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) was cultured in DMEM high-glucose medium containing 15% fetal bovine serum and 1% double antibody at 37°C, 5% CO ₂ , and saturated humidity, and passaged once every 2-3 days.

1.2 Establishment of HFF-1 cell photoaging model

HFF-1 cells of the 3rd to 5th generations with strong proliferation ability and good growth status were selected and divided into UV irradiation group, sEVs group and sIVs group to establish the HFF-1 cell photoaging model. The specific operation was as follows: when the cell density of each group reached 50% to 60%, the cell culture medium was removed and a thin layer of PBS was spread. The irradiation dose was 3J UVA+300mJ UVB/ ^cm2 . The cells were placed 10cm below the preheated UV lamp, and the probe of the UV irradiator was placed to observe the irradiation dose in real time.

1.3 CCK-8 kit to detect the viability of photoaging cells

After the HFF-1 chronic photoaging model was constructed in a six-well plate, HFF-1 cells were plated in a 96-well plate at a density of 5×10 ^3. For the experimental group cells, 5μg/ml, 10μg/ml, and 20μg/ml sEVs and sIVs were given 24h before illumination. After 48h of culture, 10μL of CCK-8 reagent was added to each well and placed in a 37°C incubator containing 5% CO ₂ for 1 hour. Subsequently, the absorbance value (OD value) of each well at 450nm was detected using an ELISA instrument, and the cell viability was calculated according to the formula = (OD value of the experimental group-OD value of the blank control group)/(negative control group-blank control group). The drug concentration was selected as 10ug/ml, and the cell proliferation was observed after 24h, 48h, and 72h of sIVs and sEVs treatment.

1.4 Senescence-associated-β-galactosidase (SA-β-GAL) staining

Normal and photoaged HFF-1 cells were plated in 6-well plates at a density of 1×10 ^5. For the experimental group, 10 μg/ml sEVs and sIVs were given 24 h before illumination, and SA-β-galactosidase (SA-β-GAL) staining was performed 72 h after treatment. Staining steps: Wash the cells once with PBS, then add 1 mL of staining fixative to each well and fix at room temperature for 15 minutes. Then prepare the staining working solution according to the instructions, add the working solution to each well and incubate in a 37°C incubator overnight. Then observe under a normal optical microscope.

1.5 Effects of sIVs and sEVs on the expression of P21, P53 and MMP-3 in photoaged cells at the mRNA level

Normal and photoaged HFF-1 cells were plated in 6-well plates at a density of 1×10 ⁵ , respectively. The experimental group cells were given 10 μg/ml sEVs and sIVs 24 h before illumination. After 48 h of treatment, the upper culture medium of each group of cells was removed and washed with PBS. Then, RNA of each group of cells was extracted immediately and reverse transcription was performed according to the instructions of the reverse transcription kit.

1.6 Effects of sIVs and sEVs on the expression of P21, P53, and MMP-3 in natural senescent cells at the mRNA level

HFF-1 cells were passaged to P25 for natural senescence cell experiments. Normal (P3) and naturally aged HFF-1 cells (P25) were plated in 6-well plates at a density of 1×10 ⁵ , and 10 μg/ml sEVs and sIVs were given to the experimental group cells, respectively. After 48 hours of culture, the upper culture medium of each group of cells was removed and washed with PBS, and then the RNA of each group of cells was immediately extracted and reverse transcription was performed according to the instructions of the reverse transcription kit.

1.7 CCK-8 kit to detect the vitality of natural senescent cells

Normal (P3 generation) and naturally aged HFF-1 cells were plated in 96-well plates at a density of 5×10 ³ , and 10 μg/ml sEVs and sIVs were given to the experimental group cells, respectively. After 48 h of culture, 10 μL of CCK-8 reagent was added to each well and incubated in a 37°C incubator containing 5% CO ₂ for 1 hour. Subsequently, the absorbance value (OD value) of each well at 450 nm was detected using an ELISA reader, and the cell viability was calculated according to the formula = (OD value of the experimental group - OD value of the blank control group) / (negative control group - blank control group).

2. Experimental results

2.1 Results of CCK-8 kit to detect photoaging cell viability

Irradiating fibroblast HFF-1 with ultraviolet light (UV, intensity 3J UVA combined with 300mJ UVB) can simulate light-induced cell aging. The results are shown in Figure 36. UV caused cell proliferation to slow down, but sEVs and sIVs significantly enhanced cell proliferation and reduced cell aging when incubated with cells for 48 hours and 72 hours, and showed a dose-dependent effect. The therapeutic effect of sIVs on photoaging was better than that of sEVs. ****: P < 0.0001; **: P < 0.01; *: P < 0.05

2.2 SA-β-GAL staining results

SA-β-Gal (Senescence-Associated β-Galactosidase) is a β-galactosidase, and upregulation of SA-β-Gal expression is an important marker of senescent cells. In this study, we used SA-β-Gal staining to assess cell senescence. The results are shown in Figure 37. Very few cells in the normal control group were stained blue, while most of the cells in the photoaging group were stained blue, indicating that the cells had undergone senescent changes; treatment with sIVs and sEVs significantly reduced the number of SA-β-gal positive cells, and the therapeutic effect of sIVs was better than that of sEVs.

2.3 Effects of sIVs and sEVs on the expression of p21, p53, and MMP-3 in photoaged cells at the mRNA level

Cellular senescence is a physiological phenotype of permanent cell cycle arrest, and p21 and p53 are both molecules related to cell cycle arrest. UV irradiation leads to the production of reactive oxygen free radicals (ROS), upregulating the expression of MMPs and leading to the degradation of matrix collagen. The results are shown in Figure 38. UV irradiation increased the mRNA expression of p21, p53 and MMP-3. However, sIVs and sEVs treatment significantly reduced the transcription levels of p21, p53 and MMP-3, which are important marker components of skin aging.

2.4 Effects of sIVs and sEVs on the expression of p21, p53, and MMP-3 in natural senescent cells at the mRNA level

After multiple passages, the cells underwent natural senescence, and the natural senescent cells also overexpressed cell cycle arrest-related molecules. The results are shown in Figure 39. sIVs and sEVs treatment significantly reduced the transcription levels of p21, p53 and MMP-3 in natural senescent cells.

2.5 Results of CCK-8 kit to detect the viability of natural senescent cells

After multiple passages, the cells naturally aged and their proliferation capacity decreased. The proliferation capacity of naturally aged cells was observed by adding two vesicles. The results are shown in Figure 40. sIVs can significantly enhance the proliferation capacity of naturally aged cells and slow down cell aging.

Example 9: Anti-aging effect of sIVs in vivo

1. Experimental methods

1.1 Establishment of in vivo photoaging model

Minimal erythema dose (MED) determination: 6-week-old Kunming (KM) female mice were shaved with an electric shaver to remove about 2×3 cm ² of hair on the back of the mice, and then a depilatory cream was used to remove the hair on the back of the mice. During each irradiation, the back of the mice was ensured to be free of hair and the back skin of each mouse was fully and evenly exposed to ultraviolet light. The ultraviolet irradiation equipment was determined according to the preliminary experiment: two UVA lamps and one UVB lamp were placed alternately in the clean bench, that is, UVA-UVB-UVA, to simulate the ultraviolet irradiation environment. After the ultraviolet lamp was turned on and stabilized for 30 minutes, the ultraviolet irradiation intensity was measured using an ultraviolet irradiator. The UVA and UVB irradiation intensities were 0.495mW/cm ² and 0.25mW/cm ² , respectively. The mice were placed in a homemade ultraviolet irradiation box to ensure that the back skin of each mouse was fully and evenly exposed to ultraviolet light. The distance between the lamp and the back of the mouse was 30cm. The minimum erythema dose of mice was determined to be UVA 1J/cm ² and UVB 0.1J/cm ² . The experimental period was 9 weeks. The initial intensity of ultraviolet light in the first week was the minimum erythema dose (MED), and the radiation dose (UVB 0.1J/cm ² , UVA 1J/cm ² ) was increased by 0.5 MED every week until the 4th week, that is, the UVB and UVA doses were 0.25 and 2.5J/cm ² respectively in the 4th week. From the 5th week onwards, the radiation dose was constant, UVB was 0.25J/cm ² , and UVA was 2.5J/cm ² .

Five days after the successful establishment of the KM mouse photoaging model, the back skin of the mouse was dried and disinfected with 75% alcohol solution. A 1mL syringe was connected to a 22G needle to inject the above-mentioned mesenchymal stem cell nanovesicle suspension (200ug/500ul) of the treatment group and PBS (500ul) of the control group into the back skin tissue of the photoaged mouse at multiple points and evenly. The injection layer was as superficial as possible. After the injection, the back skin of the mouse was gently massaged to make the injection evenly spread in the back skin tissue of the mouse. Injection was performed once every other day for a total of two weeks.

1.2 HE staining

After the treatment, the mice were killed and dissected, and the back skin tissue was extracted. Some of them were frozen and sliced to observe the fluorescence color development; some were fixed with formaldehyde and embedded in paraffin, and the slices were stained with HE. The slices were dewaxed with xylene, and the dewaxed tissue slices were placed in hematoxylin solution for 5 minutes after high-concentration alcohol to low-concentration, and finally in distilled water. After color separation in acid and alkali for a few seconds, they were rinsed with running water for 1 hour, and then dehydrated in 70% and 90% concentration alcohol for 10 minutes, and finally stained with eosin for 3 minutes. After staining, they were dehydrated with pure alcohol, and then xylene was used to make them transparent, and the slices were sealed with gum. The thickness of the epidermis and dermis of each group of mice was observed under a microscope.

1.3 Masson staining

The skin tissue of the irradiated area of the mouse was removed, and the excess tissue was quickly fixed, embedded, and sliced. The slices were treated with gradient ethanol solutions, and the staining solution and color separation solution were added according to the instructions of the Masson trichrome staining kit. The slices were then dehydrated, transparentized, and sealed for observation under a microscope.

1.4 Cell apoptosis detection

Apoptosis was detected by TdT-mediated d UTP Nick-End Labeling (TUNEL). After embedding, the skin tissue was sliced into 5 μm sections, fixed with 4% paraformaldehyde, and permeabilized with 0.5% triton-100. Proteinase K working solution and TUNEL reaction solution were added according to the instructions of the TUNEL kit. After washing, the nucleus was stained with DAPI, and the sections were sealed with anti-fluorescence quencher and observed under a confocal microscope.

1.5 β-galactosidase staining of frozen sections

After washing the frozen sections with PBS, the cells were fixed in 4% paraformaldehyde (4% PFA) at room temperature for 10 min, blocked at room temperature for 1 h, β-galactosidase antibody was added at a ratio of 100:1, and the sections were sealed after staining with DAPI and observed under a confocal microscope.

2. Experimental results

2.1 HE staining results

After UV irradiation of mouse skin, local photoaging occurred. The results are shown in Figure 41. Compared with the normal group, the epidermal thickness of the model group increased, the dermis became thinner and inflammatory cells infiltrated, indicating that the photoaging model was successfully established. The skin conditions of the UV+sIVs and UV+sEVs groups were significantly alleviated compared with the UV-PBS group, the skin structure was intact, the thickness of the epidermis was significantly thinner, the boundary with the dermis was obvious, and the inflammatory cell infiltration of the dermis was reduced.

2.2 Masson staining results

Masson staining showed the tissue arrangement in the skin. The results are shown in Figure 42. In the normal group, the collagen fibers in the dermis were densely arranged, showing wavy or bundled shapes; in the UV group, the collagen fibers in the dermis were unevenly dense, block-shaped and irregularly arranged; the sIVs group and sEVs group were improved compared with the model group, with increased and neatly arranged collagen fibers in the dermis, and the sIVs group had a significantly better repair effect on photoaged skin than sEVs.

2.3 Cell apoptosis detection results

UV irradiation of mouse skin caused apoptosis of local cells. TUNEL was used to mark apoptosis signals in tissues. The results are shown in Figure 43. TUNEL staining showed that the normal group had only a very small amount of fluorescent signals (the brighter areas or spots in the figure: apoptosis positive signals), while the UV group had widely distributed The bright fluorescence signal intensities of the sIVs group and sEVs group were reduced compared with the model group, and the inhibitory effect of sIVs on apoptosis was significantly better than that of sEVs.

2.4 Results of β-galactosidase staining of frozen sections

One of the most widely used biomarkers for senescence and senescent cells is senescence-related β-galactosidase. The results are shown in Figure 44. There was almost no bright fluorescence signal area in the skin tissue of mice in the normal control group, while the full-thickness skin tissue of the PBS group treated with UV showed high fluorescence intensity (the contrast between the positive signal and the background was obvious), and the positive signal range was wider. After treatment with sIVs and sEVs, the fluorescence intensity was reduced, and most of them were located in the surface skin tissue.

Example 10: In vivo penetration test of sIVs

1. Experimental methods

1.1 Treatment of experimental animals

In the experiment, 6-week-old female Kunming mice were used. First, an electric shaver was used to shave about 2× ^3cm2 of hair on the back of the mice, and then a depilatory cream was used to remove the hair on the back of the mice. The experiment was divided into two groups, sEVs and sIVs. Each group of mice was subcutaneously injected and topically applied with 500ul of 100ug/ml DID-labeled sEVs and sIVs (single administration).

The preparation steps of DID-labeled sEVs and sIVs are as follows: 2.5ul of DID staining solution (1ug/ul) was added to 500ul of sIVs and sEVs solutions, respectively, and incubated at 37°C in the dark for 30 minutes. After incubation, the suspension of DID-sEVs and DID-sIVs was added to an ultrafiltration tube and centrifuged at 14000g for 15 minutes. After centrifugation, 500ul of PBS was added to resuspend and centrifuged again at 14000g for 15 minutes. Subsequently, the inverted ultrafiltration tube with 500ul of PBS was centrifuged at 1000g for 2 minutes.

1.2 Slice preparation

At 2 hours, 4 hours, and 6 hours after treatment, animal skin samples were removed and cut into strips of approximately 0.5 cm in width from head to tail. These skin tissue strips were placed in OCT embedding medium to ensure complete immersion and then wrapped in aluminum foil. The samples were frozen in liquid nitrogen and then sliced or stored at -80°C. DAPI staining was used to stain the cell nucleus and anti-fluorescence quenching solution was used to seal the slides.

1.3 Determination of skin oxidative stress and inflammatory markers

The skin tissue was ground in physiological saline to prepare a homogenate, then centrifuged at 3000 r/min for 15 min at 4°C, and the supernatant was collected and stored at -80°C. The levels of oxidative stress indicators (SOD, GSH, and MDA) and inflammatory indicators (IL-6) in skin tissue were measured according to the instructions of the kit.

1.4 ELISA method to detect the content of MMP-1 and MMP-3 in skin tissue

The skin tissue was weighed, and PBS was added at a weight-volume ratio of 1 g:9 mL. The tissue was fully homogenized using an ultrasonic disruptor and centrifuged at 3000 rpm for 10 min at 4°C. The ELISA kit was operated according to the instructions, and the absorbance value of each well was measured at 450 nm. Finally, the content of MMP-3 and MMP-1 in each tissue was calculated based on the standard curve and protein concentration.

2. Experimental results

2.1 Frozen sections

In order to observe the effect of sEVs and sIVs penetrating skin tissue, equal amounts of sEVs and sIVs marked with red dye were applied to the back skin tissue, and the results are shown in Figure 45. Within 2 hours after application, a small amount of both penetrated the epidermis to reach the dermis (weak fluorescence signal); 4 hours after external application, the dermis showed a brighter area and the fluorescence signal was enhanced, indicating that most of the drugs could reach the dermis; and 6 hours after external application, the drug could be distributed throughout the skin layer, and the whole layer of the skin showed a bright fluorescence signal. The position reached by sIVs was farther than that of sEVs and was more evenly distributed throughout the skin. On the other hand, after sEVs and sIVs were treated by subcutaneous injection, a large number of vesicle signals could be observed in the dermis of both groups of skin 2 hours after injection; vesicle signals were observed in the epidermis 4 hours after injection, and the sIVs signal was stronger and distributed farther; 6 hours after injection, sIVs was more strongly and widely distributed throughout the skin. In summary, the speed and efficiency of sIVs reaching the dermis after application or percutaneous injection were significantly better than sEVs.

2.2 Results of determination of skin oxidative stress and inflammatory indicators

Oxidative damage is one of the main factors of skin aging. The ability of aging skin to scavenge reactive oxygen species (ROS) decreases, which causes skin oxidative stress and leads to skin aging. Improving the antioxidant capacity of the skin is the main way to fight aging. Various antioxidant enzymes such as superoxide dismutase (SOD) and reduced glutathione (GSH) reduce oxidative stress by eliminating ROS and reduce the generation of the oxidation product MDA. The results are shown in Figure 46. Compared with the normal group, the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the skin tissue of mice in the UV group were reduced, and the level of lipid peroxidation product MDA was increased (P<0.05). Compared with the UV group, the levels of SOD and reduced GSH in the skin tissue of mice in the UV+sIVs group and UV+sEVs group were increased, and the level of MDA was decreased (P<0.05). The results of inflammatory factors showed that the IL-6 level of mice in the model group was increased, and the level of IL-6 inflammatory factor decreased after treatment with sIVs and sEVs. This shows that sIVs can reduce skin oxidative stress and reduce skin inflammatory response.

2.3 Detection results of MMP-1 and MMP-3 content in skin tissue

MMP-1 and MMP-3 are two important matrix metalloproteinases, which are expressed more in aged skin. MMP-1 and MMP-3 can reduce collagen and induce skin aging. The expression of skin aging-related factors increased after UV irradiation, and the results are shown in Figure 47. The levels of MMP-1 and MMP-3 in the back skin tissue of mice in the UV treatment group increased, and after treatment with sIVs and sEVs, the levels of MMP-1 and MMP-3 decreased.

Example 11: Anti-aging study of sIVs derived from adipose-derived mesenchymal stem cells

1. Experimental methods

1.1 Isolation and extraction of adipose-derived mesenchymal stem cells

Six-week-old male C57/BL6 mice were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital and then the inguinal fat pad was removed. Adipose mesenchymal stem cells were successfully isolated by mincing the adipose tissue and rinsing it with PBS, followed by digestion with 1 mg/mL type I collagenase at 37°C for 40 minutes. The cells were resuspended in DMEM by centrifugation, filtered through a 70 μm nylon filter, inoculated in DMEM containing 10% fetal bovine serum, 100 U/mL streptomycin and 100 U/mL penicillin, and cultured in a 37°C, 5% CO ₂ incubator. Once the cells reached 80% density, they were subcultured.

1.2 Preparation of adipose-derived mesenchymal stem cells-derived sIVs (ADSCs-sIVs)

Referring to the method of Example 2, sIVs and sEVs derived from adipose mesenchymal stem cells were prepared.

1.3 Effects of sIVs derived from adipose-derived mesenchymal stem cells on reactive oxygen species in photoaged cells

After the HFF-1 chronic photoaging model was constructed in a six-well plate, HFF-1 cells were plated in a 96-well plate at a density of 5×10 ^3. For the experimental group, 10 μg/ml sEVs and sIVs derived from adipose mesenchymal stem cells were given 24 hours before illumination, and superoxide anion fluorescence detection was performed after 48 hours of culture. The level of superoxide anion in cells was detected using a superoxide anion fluorescence detection probe (Biyuntian: s0063): Dihydroethidium (DHE) is taken up by cells, and ethidium is produced under the action of superoxide anions, which then binds to RNA or DNA to produce red fluorescence. The product after combining with hydroxyl (—OH) will cause cell DNA damage. Usually, an appropriate solution containing 0.5-5 μM DHE is incubated with cells at 37°C for about 30 minutes to load the fluorescent probe, which can then be properly washed and detected using a flow cytometer or other fluorescence detection instrument.

1.4 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the proliferation of photoaged cells

After the HFF-1 chronic photoaging model was constructed in a six-well plate, HFF-1 cells were plated in a 96-well plate at a density of 5×10 ^3. The experimental group cells were given 10 μg/ml sEVs and sIVs derived from adipose mesenchymal stem cells 24 hours before light exposure, and CCK-8 detection was performed after 48 hours of culture, using the same method as above. The detection method is in accordance with Section 1.3.

1.5 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the proliferation of natural senescent cells

HFF-1 cells were serially passaged to the senescent state P25. Senescent HFF-1 cells were plated in a 96-well plate at a density of 5×10 ³ , and the experimental group cells were given 10 μg/ml sEVs and sIVs derived from adipose mesenchymal stem cells, and CCK-8 detection was performed after 48 hours of culture, using the same method as above.

1.6 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the anti-aging ability of photoaged cells

The senescence-associated-β-galactosidase (SA-β-GAL) staining method refers to Section 1.4 of Example 8.

1.7 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the antioxidant capacity of skin tissue in photoaged mice.

The construction of the photoaging model and frozen sections refer to Example 9. Five days after the successful establishment of the KM mouse photoaging model, the back skin of the mouse was dried and disinfected with 75% alcohol solution. A 1mL syringe was connected to a 22G needle to inject sIVs (ADSCs-sIVs) and sEVs (ADSCs-sEVs) (200ug/500ul) derived from adipose mesenchymal stem cells and PBS (500ul) of the control group into the back skin tissue of the photoaged mouse at multiple points and evenly. The injection layer was as superficial as possible. After the injection, the back skin of the mouse was gently massaged to make the injection evenly spread in the back skin tissue of the mouse. Inject once every other day for a total of two weeks. After the treatment, superoxide anion fluorescent probe detection was performed according to the instructions.

2. Experimental results

2.1 Effects of sIVs derived from adipose-derived mesenchymal stem cells on reactive oxygen species in photoaged cells

DHE can be dehydrogenated under the action of superoxide anions in cells to produce ethidium (such as ethidium bromide), which is one of the most commonly used superoxide anion fluorescence detection probes. When the level of superoxide anions in cells is high, more ethidium is produced, and the fluorescence signal is stronger, which can be used to determine the amount and changes of cellular ROS content. The results are shown in Figure 48. The fluorescence brightness and range of the sEVs and sIVs groups derived from adipose mesenchymal stem cells were significantly weaker than those of the untreated UV irradiated group, indicating that sEVs and sIVs derived from adipose mesenchymal stem cells can also significantly reduce the superoxide anion level of cells after UV irradiation, improve intracellular reactive oxygen species, and reduce oxidative stress, thereby reducing damage to cell DNA, and ADSCs-sIVs are better than ADSCs-sEVs.

2.2 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the proliferation of photoaged cells

The results are shown in Figure 49. Both sEVs and sIVs derived from adipose-derived mesenchymal stem cells showed the ability to enhance the proliferation of photoaged cells.

2.3 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the proliferation capacity of natural senescent cells

The results are shown in Figure 50. sEVs and sIVs derived from adipose-derived mesenchymal stem cells also showed that they could enhance the proliferation capacity of naturally senescent cells.

2.4 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the anti-aging ability of photoaged cells

The results are shown in Figure 51. Treatment with sIVs and sEVs derived from adipose-derived mesenchymal stem cells can also significantly reduce the number of SA-β-gal-positive cells, and the therapeutic effect of sIVs is better than that of sEVs.

2.5 Effect of sIVs derived from adipose-derived mesenchymal stem cells on the antioxidant capacity of skin tissue of photoaged mice.

The results are shown in Figure 52. There is almost no fluorescence signal in the skin tissue of mice in the normal control group, while the full-thickness skin of the PBS group after UV treatment shows large bright areas or spots, and the dermis shows high fluorescence intensity, which indicates that the ROS signal of the skin of photoaged mice is enhanced and there is severe oxidative stress. The fluorescence intensity was weakened after treatment with ADSCs-sIVs and ADSCs-sEVs, indicating that the oxidative stress was improved. sEVs and sIVs derived from adipose mesenchymal stem cells can also significantly reduce the level of superoxide anion in the back skin tissue of mice after UV irradiation, improve intracellular reactive oxygen species, and reduce oxidative stress, thereby reducing damage to cell DNA, and ADSCs-sIVs are better than ADSCs-sEVs.

Example 12: Anti-aging study of sIVs derived from bone marrow mesenchymal stem cells

1. Experimental methods

1.1 Isolation and extraction of bone marrow mesenchymal stem cells

Extraction of BMSCs from SD rats using whole bone marrow adherence method

(1) Three-week-old SD rats were killed by cervical dislocation and then immersed in 75% alcohol for 5 minutes.

(2) Open the sterile dressing bag in the clean bench and place the disinfected rat in the sterile dressing bag. Cut the epidermis and subcutaneous tissue, separate the femur and tibia of the SD rat and remove them completely. Then use gauze to remove the muscles and other soft tissues attached to the femur and tibia, and place the removed bones in a 60 mm culture dish.

(3) Add 75% alcohol to the culture dish and soak for 3 minutes. Then transfer the bones to a new culture dish and rinse off the residual alcohol with PBS.

(4) Use ophthalmic forceps to clamp the bone and ophthalmic scissors to cut off the epiphysis at both ends of the long bone to expose the medullary cavity. Use a 1 ml syringe to absorb DMEM complete culture medium containing a mixture of 10% FBS and 1% penicillin-streptomycin to repeatedly rinse the medullary cavity and collect the culture medium containing cells into a new culture dish.

(5) The culture medium containing the cells is inoculated into a T25 culture flask at an appropriate density and labeled as primary cells (M0).

(6) The cell culture incubator was set to 37°C with 5% CO ₂ , and the culture flask was placed therein for static culture.

1.2 Effects of sEVs (BMSCs-sEVs) and sIVs (BMSCs-sIVs) derived from bone marrow mesenchymal stem cells on the proliferation ability of photoaged cells: After constructing the HFF-1 chronic photoaging model in a six-well plate, HFF-1 cells were plated in a 96-well plate at a density of 5×10 ^3. The experimental group cells were given 10 μg/ml sEVs and sIVs derived from bone marrow mesenchymal stem cells 24 hours before illumination, and CCK-8 detection was performed after 48 hours of culture, using the same method as above.

1.3 Effect of sIVs derived from bone marrow mesenchymal stem cells on the proliferation capacity of natural senescent cells

HFF-1 cells were serially passaged to the senescent state P25. Senescent HFF-1 cells were plated in a 96-well plate at a density of 5×10 ³ , and the experimental group cells were given 10 μg/ml sEVs and sIVs derived from bone marrow mesenchymal stem cells, and CCK-8 detection was performed after 48 hours of culture, using the same method as above.

1.4 Effects of sEVs and sIVs derived from bone marrow mesenchymal stem cells on the levels of superoxide dismutase (SOD) and reduced glutathione in photoaged cells: HFF-1 cells were seeded at a density of 1×10 ⁵ in six-well plates. After the cells adhered to the wall, 10 ug/ml of sIVs and sEVs were added respectively. After UV irradiation, SOD and reduced GSH were detected according to the instructions.

1.5 Effects of sIVs and sEVs derived from bone marrow mesenchymal stem cells on the contents of MMP-1 and MMP-3 in the skin tissue of photoaged mice.

The construction of the photoaging model and frozen sections refer to Example 9. Five days after the successful establishment of the KM mouse photoaging model, the back skin of the mouse was dried and disinfected with 75% alcohol solution. A 1mL syringe was connected to a 22G needle, and sIVs and sEVs (200ug/500ul) derived from bone marrow mesenchymal stem cells and PBS (500ul) of the control group were injected into the back skin tissue of the photoaged mouse at multiple points and evenly. The injection layer was as superficial as possible. After the injection, the back skin of the mouse was gently massaged to make the injection evenly spread in the back skin tissue of the mouse. Inject once every other day for a total of two weeks of treatment. After the treatment, the MMP-1 and MMP-3 instructions were followed for detection.

1.6 Effects of sIVs and sEVs derived from bone marrow mesenchymal stem cells on the expression of β-galactosidase in the skin tissue of photoaged mice.

After washing the frozen sections with PBS, the cells were fixed in 4% paraformaldehyde (4% PFA) (RT) for 10 min at room temperature, blocked for 1 h at room temperature, and β-galactosidase antibody was added at a ratio of 100:1 overnight, and the sections were sealed after staining with DAPI and observed under a confocal microscope.

2. Experimental results

2.1 Effects of sEVs and sIVs derived from bone marrow mesenchymal stem cells on the proliferation of photoaged cells

The proliferation ability of aged cells will be weakened. As shown in Figure 53, both sEVs and sIVs derived from bone marrow mesenchymal stem cells showed that they could enhance the proliferation ability of photoaged cells, and the ability of BMSCs-sIVs to promote the proliferation of photoaged cells was better than that of BMSCs-sEVs.

2.2 Effect of sIVs derived from bone marrow mesenchymal stem cells on the proliferation ability of naturally aged HFF-1 cells

The results are shown in Figure 54. Both sEVs and sIVs derived from bone marrow mesenchymal stem cells showed the ability to enhance the proliferation of photoaged cells.

2.3 Effects of sEVs and sIVs derived from bone marrow mesenchymal stem cells on superoxide dismutase (SOD) and reduced glutathione (GSH) levels in photoaged cells.

Oxidative damage is one of the main factors of skin aging. The ability of aging skin to scavenge reactive oxygen species (ROS) decreases, which causes skin oxidative stress and leads to skin aging. Various antioxidant enzymes such as superoxide dismutase (SOD) and reduced glutathione (GSH) reduce oxidative stress by eliminating ROS and reduce the generation of the oxidation product MDA. The results are shown in Figure 55. Compared with the UV group, the levels of SOD and reduced GSH in the skin tissues of mice in the BMSCs-sIVs treatment group and the BMSCs-sEVs group were increased, and BMSCs-sIVs could more significantly increase the level of GSH in cells than BMSCs-sEVs.

2.4 Effects of sIVs and sEVs derived from bone marrow mesenchymal stem cells on the levels of MMP-1 and MMP-3 in the skin tissue of photoaged mice.

MMP-1 and MMP-3 are two important matrix metalloproteinases, which are expressed more in aging skin. MMP-1 and MMP-3 can reduce collagen and induce skin aging. The results are shown in Figure 56. The levels of MMP-1 and MMP- in the back skin tissue of mice in the UV treatment group increased, and after treatment with BMSCs-sIVs and BMSCs-sEVs, the levels of MMP-1 and MMP-3 decreased.

2.5 Effects of sIVs and sEVs derived from bone marrow mesenchymal stem cells on the expression of β-galactosidase in the skin tissue of photoaged mice.

One of the most widely used biomarkers of senescence and senescent cells is senescence-related β-galactosidase. The results are shown in Figure 57. Compared with the normal control group mice, the PBS group treated with UV showed extensive bright area patches in the full-thickness skin tissue, and the fluorescence signal was the strongest. After treatment with BMSCs-sIVs and BMSCs-sEVs, the brightness and distribution range of the fluorescence signal decreased, and the fluorescence intensity of the BMSCs-sIVs group was lower than that of BMSCs-sEVs, indicating that the expression of senescence-related β-galactosidase in the skin tissue of mice in the BMSCs-sIVs group was significantly reduced.

Combined with Examples 8-12, it can be seen that sIVs derived from umbilical cord mesenchymal stem cells, sIVs derived from adipose mesenchymal stem cells, and sIVs derived from bone marrow mesenchymal stem cells can effectively improve the proliferation activity of human skin fibroblasts, improve cell aging (including intrinsic aging (natural aging) and exogenous aging (such as photoaging)), and at the same time improve UV-induced oxidative stress and aging manifestations in mouse skin.

Example 13: Study on hair follicle regeneration using sIVs

1. Experimental Animals

The experimental animals were healthy male 7-week-old C57BL/6J mice purchased from Beijing Weitonglihua Company and housed in a room (23±2℃, 12h light-dark alternation) Independent ventilation cage SPF system.

2. Experimental methods

2.1 Construction of a mouse model of hair follicle regeneration induced by hair plucking:

All mice were randomly divided into 3 groups. After anesthesia with Shutai plus xylazine hydrochloride, low melting point paraffin was melted and evenly applied to the back of the mice. The back hair was plucked after solidification. The PBS solvent control group was intradermally injected with 400ul PBS solution, the sIVs group was intradermally injected with the same volume of sIVs solution with a concentration of 500ug/ml at multiple points, and the sEVs group was intradermally injected with the same volume of sEVs solution with a concentration of 500ug/ml, once every other day for one week; the dynamic changes of hair growth in the plucked area of the mice were recorded by taking pictures. The completion of hair removal was recorded as 0d after hair removal.

2.2 Frozen sections and immunofluorescence staining

Preparation of frozen sections: After the treatment, the skin of the animal was sampled, and the skin tissue was cut into long strips with a width of about 0.5 cm from head to tail, placed longitudinally in OCT embedding medium and fully immersed, wrapped with aluminum foil, and sliced or stored at -80°C after liquid nitrogen freezing. The frozen sections were dried at room temperature for about 10 minutes and rinsed with PBS; 4% PFA + 4% sucrose was added to the tissue section samples and fixed at room temperature for 15 minutes; the fixative was discarded, and the cells were perforated with 0.2% Trixton X-100 for about 10 minutes, and rinsed with PBS three times, each time for 5 minutes; 2% BSA + 5% sheep serum (100ml + 2gBSA + 5ml sheep serum) was blocked at room temperature for 30 minutes. Reagents: recombinant Anti-Ki67 antibody (Abcam; ab16667); recombinant Anti-beta Catenin antibody (Abcam; ab32572).

2.3 Extraction of total protein from skin tissue and detection of β-catenin expression level in skin tissue by Western blot: Weigh a certain amount of skin tissue in a centrifuge tube, cut it into pieces, add the corresponding volume of RIPA lysis buffer at a ratio of 1:10 (tissue mg: RIPA μL), and add the corresponding PMSF and phosphatase inhibitor at a ratio of 1:100 (PMSF/protease inhibitor/phosphatase inhibitor: RIPA) to prevent protein degradation and dephosphorylation in the tissue. Then add steel beads, grind on a high-pass tissue grinder, and place on ice for lysis for 30 minutes. Centrifuge at 10000r/min, 10min, 4℃ and take the supernatant to obtain the total protein extract. The total protein content was detected using the BCA protein quantitative analysis kit for subsequent detection. Take the above total protein extract and heat it at 100℃ for 10min to denature. Then, electrophoresis was performed using 10% separation gel at 160V for 50min, wet transfer was performed at 200mA for 80min, and the samples were blocked with 5% skimmed milk powder for 2h (5% bovine serum albumin (BSA) was used to detect phosphorylated proteins). The primary antibody was incubated on a shaker at 4°C overnight, the secondary antibody was incubated at room temperature for 1h, and finally exposed to light for development.

2.4 HE staining to detect changes in hair follicle morphology

Paraffin sections were dewaxed and hydrated, incubated with hematoxylin for 1-5 min and bluing, incubated with eosin for 30 s-3 min, washed thoroughly with double distilled water, dehydrated and made transparent, sealed with neutral gum, and images were collected using a microscope.

3. Experimental results

3.1 Treatment results of the mouse model of hair follicle regeneration induced by hair plucking

The results are shown in Figure 58. On the 7th and 14th days after hair plucking, the changes in skin color and hair growth of the mice at the hair plucking site were recorded. The results showed that the skin color changes and hair growth of the mice in the sEVs group and sIVs group were significantly faster than those in the PBS group, and the hair growth of the mice in the sIVs group was significantly faster than that in the sEVs group.

3.2 Immunofluorescence staining results

The Wnt/β-catenin pathway is considered to be the main pathway involved in hair regeneration and the primary signal for hair follicle development and regeneration. High expression of β-catenin indicates that the pathway for hair follicle regeneration is activated. In addition, Ki67 is a signal for cell proliferation. The expression of Ki67 by hair follicles represents the development and regeneration of hair follicles. The immunofluorescence results are shown in Figures 59-60, indicating that the expression content of β-catenin (the range and intensity of bright fluorescent areas or spots) in the sEVs and sIVs groups was significantly higher than that in the PBS group; the expression content of Ki67, a hair follicle proliferation-related factor, in the sEVs and sIVs groups was significantly higher than that in the PBS group, and the sIVs group could significantly increase the expression level of Ki67 compared with sEVs.

3.3 WB results

β-catenin is considered to be the initial signal involved in hair regeneration. The β-catenin signal of the tissue was further detected using a weter blot. The results are shown in Figure 61. Compared with the PBS group, the expression of β-catenin in the back skin tissue of mice in the sEVs group and the sIVs group was significantly increased, and the sIVs group promoted the expression of β-catenin signal to a higher extent than sEVs.

3.4 HE staining results

After depilation of mice, during the process of hair regeneration, the number of hair follicles increases and the hair bulb expands to support hair growth. As shown in Figure 62, the hair follicles in the depilatory PBS treatment group (normal control) were fewer, the hair bulb diameter was thinner, and the skin was thinner. However, after treatment with sIVs and sEVs, on the 14th day, the number of skin hair follicles, skin thickness, and hair bulb diameter of mice in the sEVs and sIVs groups were significantly higher than those in the PBS group, and the sIVs treatment group could significantly increase the hair bulb diameter compared to sEVs.

In summary, the sIVs prepared by the present invention can promote the development and regeneration of hair follicles, promote cell proliferation, and help regenerate more hair. It is expected to be used for the prevention and treatment of hair loss (such as senile alopecia, alopecia areata, chemotherapy-induced alopecia, seborrheic alopecia, etc.).

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

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

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

Claims (15)

The invention discloses an application of intracellular nanovesicles derived from mesenchymal stem cells in preparing products for skin anti-aging, photo-aging skin repair or promoting hair follicle regeneration. A use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and/or treating diseases or hair loss caused by photoaging. The use according to claim 1 or 2, characterized in that the vesicle is prepared by a method comprising the following steps: (1) dispersing mesenchymal stem cells in a suspension solvent and subjecting them to ultrasonic treatment; (2) centrifuging the liquid obtained in step (1) once or multiple times to obtain the supernatant; (3) The supernatant obtained in step (2) is subjected to ultracentrifugation to obtain a precipitate. The use according to claim 3, characterized in that the amplitude of the ultrasonic treatment in step (1) is 20%-25%, and/or the time of the ultrasonic treatment is 10-20s. The application as described in claim 3 is characterized in that the amplitude of the ultrasonic treatment in step (1) is 20%; and/or the time of the ultrasonic treatment is 15s, on 2s, off 2s. The use according to claim 3, characterized in that the number of centrifugal treatments in step (2) is two, and their respective parameters are: 1000-3000g, 5-20 minutes; 10000-30000g, 20-40 minutes; and/or, The parameters of the ultracentrifugation treatment in step (3) include 100000-180000g, 50-100 minutes. The use according to claim 1 or 2, characterized in that the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells. The use according to claim 1 or 2, characterized in that the average particle size of the vesicles is 50-100 nm. The use according to claim 1 is characterized in that the product is a cosmetic or medical beauty product, which is used on the skin or skin appendages. The use according to claim 9, characterized in that the product further comprises excipients acceptable in the field of cosmetics or medical beauty products. The use according to claim 1, characterized in that the product is an injection or a transdermal preparation. The use according to claim 2, characterized in that the drug is an injection or a transdermal preparation. The use according to any one of claims 1, 3-11, characterized in that the skin is located on the face, neck, hands, arms, back, legs, abdomen or any combination thereof of the subject, preferably on the face. The use according to any one of claims 1 and 3 to 11, characterized in that the aging includes endogenous aging and exogenous aging, in particular photoaging. The use according to any one of claims 1 and 3 to 11, characterized in that the vesicles are administered to the subject by injection, topically or transdermally.