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WO2025067020A1 - Utilisation de nanovésicule intracellulaire dérivée de cellules souches adultes dans l'anti-vieillissement et la régénération de follicules pileux - Google Patents

Utilisation de nanovésicule intracellulaire dérivée de cellules souches adultes dans l'anti-vieillissement et la régénération de follicules pileux Download PDF

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WO2025067020A1
WO2025067020A1 PCT/CN2024/119771 CN2024119771W WO2025067020A1 WO 2025067020 A1 WO2025067020 A1 WO 2025067020A1 CN 2024119771 W CN2024119771 W CN 2024119771W WO 2025067020 A1 WO2025067020 A1 WO 2025067020A1
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sivs
sevs
cells
skin
aging
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Chinese (zh)
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张晓敏
张咪
张慧
李筱荣
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TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
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TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • 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.
  • 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.).
  • 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 wrinkle
  • 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.
  • drugs for hair loss treatment include retinoic acid, atropine, glucocorticoids, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • IVs intracellular vesicles
  • 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.
  • 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.
  • the vesicles are intracellular nanovesicles (small intracellular vesicles, sIVs).
  • 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).
  • 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).
  • UC-MSC umbilical cord mesenchymal stem cells
  • BM-MSC bone marrow mesenchymal stem cells
  • AD-MSC adipose mesenchymal stem cells
  • dental pulp mesenchymal stem cells placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).
  • 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.
  • 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.
  • mammal includes rats, mice, guinea pigs, rabbits, dogs, monkeys, chimpanzees, humans, etc., especially humans.
  • 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.
  • prevent refers to avoiding, minimizing or making the onset or development of a disease difficult by treating it before it occurs.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • 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).
  • 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.
  • 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.
  • sEVs small extracellular vesicles
  • Example 2 the optimization process of the separation parameters of intracellular nanovesicles is shown in FIG2 .
  • NTA Nanoparticle Tracking Analysis
  • 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.
  • FIG 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
  • NTA Nanoparticle Tracking Analysis 3.3Dev Build 3.3.104 for detection.
  • the camera mode is set to sCMOS.
  • the laser type is set to Blue488, and the viscosity is set to 0.9cP.
  • the protein 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 1-4 ⁇ l of marker on each side.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • proteomic analysis 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.
  • 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.
  • Buffer A is 100% acetonitrile (ACN)
  • Buffer B is 0.1% trifluoroacetic acid (TFA).
  • the peptides treated with enzymes were loaded onto a homemade Trap column (100 ⁇ m ⁇ 2 cm, C18 filler, particle size 3 ⁇ m, 120A) using phase A (containing 0.1% formic acid, 2% acetonitrile and 97.9% water) at a flow rate of 3 ⁇ l/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 ⁇ m ⁇ 15 cm, C18 filler, particle size 1.9 ⁇ m, 120A) to form an electrically charged spray and finally entered the mass spectrometer detector.
  • phase A containing 0.1% formic acid, 2% acetonitrile and 97.9% water
  • phase B containing 97.9% acetonitrile, 2% water and 0.1% formic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CC cell component
  • MF molecular function
  • BP biological process
  • 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.
  • Collect sEVs and sIVs of MSCs adjust to equal mass, and operate according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit.
  • ELISA enzyme-linked immunosorbent assay
  • Use an enzyme reader to read the absorbance value of each well in the well plate, using a wavelength of 450nm/540nm.
  • 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.
  • sIVs contained higher levels of intracellular organelle proteins than sEVs ( Figure 18).
  • sIVs showed increased expression levels of proteins associated with membrane-enriched organelles such as endosomes, endoplasmic reticulum, and Golgi apparatus.
  • sEVs contained more abundant cellular membrane proteins ( Figure 18). This suggests that sIVs have intracellular characteristics.
  • 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.
  • organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways.
  • sEVs and sIVs 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.
  • 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).
  • CC cellular components
  • BP biological process
  • 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.
  • 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-1 ⁇ (IL-1 ⁇ ) 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 ⁇ (TNF ⁇ ) ( Figure 23E) in sIVs was lower.
  • 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.
  • sIVs play a key role in the intracellular material transport process, especially mediating the exchange of substances between organelles.
  • 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.
  • Example 6 sIVs have unique miRNA expression profiles
  • RNA concentration was determined using Measurements were performed using the Qubit TM RNA Assay Kit in a Flurometer. Detection was performed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 System.
  • 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 ⁇ L elution buffer. Finally, library quality was assessed on an Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.
  • 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.
  • the raw data (raw reads) in fastq format were processed by custom Perl and Python scripts.
  • clean data clean reads
  • the raw data were processed by custom Perl and Python scripts.
  • clean data clean reads
  • the Q20, Q30 and GC content of the raw data were calculated.
  • a certain length range was selected from the clean reads for all downstream analyses.
  • Bowtie Longmead 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.
  • the mapped small RNA tags were used to search for known miRNAs.
  • 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.
  • 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.
  • 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.
  • target gene candidates were GO enrichment analysis.
  • 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.
  • 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.
  • miRNAs microRNAs
  • RNA 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).
  • 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.
  • miRNAs such as miR-148a-3p, miR-21-5p and miR-100-5p were expressed at higher levels in both sEVs and sIVs ( Figure 28).
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 7 sIVs have unique lipidomic characteristics
  • centrifuged at 3000rpm centrifugal force 900 ⁇ g, radius 8.6cm
  • 15 minutes at 4°C the samples.
  • 3000rpm centrifugal force 900 ⁇ g, radius 8.6cm
  • take out 300 ⁇ L from the supernatant transfer to an EP tube, and vacuum dry.
  • Add 100 ⁇ L 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.
  • centrifuge at 13000rpm centrifugal force 16200 ⁇ g, radius 8.6cm
  • 15 minutes at 4°C take 75 ⁇ L of the supernatant and transfer it to the injection bottle, ready for machine detection.
  • Vanquish ultra-high performance liquid chromatograph was used, and Waters ACQUITY UPLC HSS T3 (2.1 mm ⁇ 100 mm, 1.8 ⁇ m) liquid chromatography column was used for chromatographic separation of the target compounds.
  • 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.
  • 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°C, 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PC, PI, PE, PG, and OxPI are significantly overexpressed in sIVs, among which PC is overexpressed 200 times in sIVs.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HFF-1 human skin fibroblasts
  • 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.
  • 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.
  • 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.
  • 5 ⁇ g/ml, 10 ⁇ g/ml, and 20 ⁇ g/ml sEVs and sIVs were given 24h before illumination.
  • 10 ⁇ L of CCK-8 reagent was added to each well and placed in a 37°C incubator containing 5% CO 2 for 1 hour.
  • the drug concentration was selected as 10ug/ml, and the cell proliferation was observed after 24h, 48h, and 72h of sIVs and sEVs treatment.
  • HFF-1 cells Normal and photoaged HFF-1 cells were plated in 6-well plates at a density of 1 ⁇ 10 5.
  • 10 ⁇ g/ml sEVs and sIVs were given 24 h before illumination, and SA- ⁇ -galactosidase (SA- ⁇ -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.
  • HFF-1 cells 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 ⁇ g/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.
  • 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 ⁇ g/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.
  • UV ultraviolet light
  • 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
  • SA- ⁇ -Gal (Senescence-Associated ⁇ -Galactosidase) is a ⁇ -galactosidase, and upregulation of SA- ⁇ -Gal expression is an important marker of senescent cells.
  • SA- ⁇ -Gal staining we used SA- ⁇ -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- ⁇ -gal positive cells, and the therapeutic effect of sIVs was better than that of sEVs.
  • 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.
  • ROS reactive oxygen free radicals
  • Figure 38 UV irradiation increased the mRNA expression of p21, p53 and MMP-3.
  • sIVs and sEVs treatment significantly reduced the transcription levels of p21, p53 and MMP-3, which are important marker components of skin aging.
  • MED Minimal erythema dose
  • 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 .
  • MED minimum erythema dose
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • TUNEL TdT-mediated d UTP Nick-End Labeling
  • the cells were fixed in 4% paraformaldehyde (4% PFA) at room temperature for 10 min, blocked at room temperature for 1 h, ⁇ -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.
  • 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.
  • mice 6-week-old female Kunming mice were used.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SOD superoxide dismutase
  • GSH reduced glutathione
  • 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.
  • 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
  • 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 ⁇ m 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.
  • ADSCs-sIVs adipose-derived mesenchymal stem cells-derived sIVs
  • sIVs and sEVs derived from adipose mesenchymal stem cells were prepared.
  • 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.
  • 10 ⁇ g/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.
  • DHE Dihydroethidium
  • ethidium 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.
  • an appropriate solution containing 0.5-5 ⁇ M 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.
  • 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 ⁇ g/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.
  • 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 ⁇ g/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.
  • SA- ⁇ -GAL staining method refers to Section 1.4 of Example 8.
  • 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.
  • 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.
  • superoxide anion fluorescent probe detection was performed according to the instructions.
  • 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.
  • ethidium such as ethidium bromide
  • 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.
  • 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
  • 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.
  • 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.
  • the culture medium containing the cells is inoculated into a T25 culture flask at an appropriate density and labeled as primary cells (M0).
  • the cell culture incubator was set to 37°C with 5% CO 2 , and the culture flask was placed therein for static culture.
  • BMSCs-sEVs sEVs
  • BMSCs-sIVs sIVs
  • 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 ⁇ g/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.
  • 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.
  • 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.
  • 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.
  • the cells were fixed in 4% paraformaldehyde (4% PFA) (RT) for 10 min at room temperature, blocked for 1 h at room temperature, and ⁇ -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.
  • 4% PFA 4% paraformaldehyde
  • 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.
  • SOD superoxide dismutase
  • GSH reduced glutathione
  • 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.
  • 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 ⁇ -galactosidase in the skin tissue of mice in the BMSCs-sIVs group was significantly reduced.
  • sIVs derived from umbilical cord 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
  • the experimental animals were healthy male 7-week-old C57BL/6J mice purchased from Beijing Weitonglihua Company and housed in a room (23 ⁇ 2°C, 12h light-dark alternation) Independent ventilation cage SPF system.
  • 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
  • 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.
  • 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).
  • 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°C 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).
  • BSA bovine serum albumin
  • 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.
  • the Wnt/ ⁇ -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 ⁇ -catenin indicates that the pathway for hair follicle regeneration is activated.
  • Ki67 is a signal for cell proliferation. The expression of Ki67 by hair follicles represents the development and regeneration of hair follicles.
  • ⁇ -catenin is considered to be the initial signal involved in hair regeneration.
  • the ⁇ -catenin signal of the tissue was further detected using a weter blot.
  • the results are shown in Figure 61.
  • the expression of ⁇ -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 ⁇ -catenin signal to a higher extent than sEVs.
  • the number of hair follicles increases and the hair bulb expands to support hair growth.
  • the hair follicles in the depilatory PBS treatment group normal control
  • the hair bulb diameter was thinner
  • the skin was thinner.
  • 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.
  • 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.).
  • hair loss such as senile alopecia, alopecia areata, chemotherapy-induced alopecia, seborrheic alopecia, etc.

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Abstract

L'invention concerne l'utilisation d'une nanovésicule intracellulaire dérivée de cellules souches adultes dans l'anti-vieillissement, la réparation et la régénération de la peau et/ou des phanères, en particulier l'utilisation d'une nanovésicule intracellulaire dérivée de cellules souches mésenchymateuses dans le vieillissement et la régénération des follicules pileux de la peau. Une telle nanovésicule intracellulaire peut réduire l'expression de molécules liées au vieillissement, améliorer l'activité de prolifération des fibroblastes de la peau humaine, améliorer l'activité SOD, améliorer efficacement le vieillissement cellulaire (y compris le vieillissement endogène [vieillissement naturel] et le vieillissement exogène [tel que le photovieillissement]), et peut également améliorer efficacement le vieillissement dans un modèle animal. De plus, la nanovésicule intracellulaire peut également favoriser le développement et la régénération du follicule pileux, faciliter la régénération de plus de cheveux, et est censée être utilisée pour prévenir et traiter l'alopécie. Par conséquent, la nanovésicule intracellulaire présente de très bonnes perspectives d'application et une très bonne valeur de recherche.
PCT/CN2024/119771 2023-09-26 2024-09-19 Utilisation de nanovésicule intracellulaire dérivée de cellules souches adultes dans l'anti-vieillissement et la régénération de follicules pileux Pending WO2025067020A1 (fr)

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WO2024260476A1 (fr) * 2023-06-21 2024-12-26 天津医科大学眼科医院 Procédé de préparation de nanovésicules d'origine intracellulaire et utilisation de nanovésicules
CN118806683B (zh) * 2023-09-26 2024-11-22 天津医科大学眼科医院 一种成体干细胞源的细胞内纳米囊泡在抗衰老和毛囊再生中的应用
CN120242070B (zh) * 2025-06-04 2025-08-22 浙江大学 协同抗衰老与成骨的干细胞源双区室囊泡及其制备与应用

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CN118806683A (zh) * 2023-09-26 2024-10-22 天津医科大学眼科医院 一种成体干细胞源的细胞内纳米囊泡在抗衰老和毛囊再生中的应用

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EP0537092A1 (fr) * 1991-10-08 1993-04-14 Patrinove Societe Civile Vecteurs vésiculaires infracellulaires et leurs applications dans les compositions cosmétiques, médicamenteuses et les milieux de culture
US20140050677A1 (en) * 2006-05-24 2014-02-20 David Michael Ott Personal care and medicinal products incorporating bound organosulfur groups
CN113286578A (zh) * 2018-08-09 2021-08-20 格林威治大学 制备脂质体的方法
WO2022066845A1 (fr) * 2020-09-23 2022-03-31 Aaron Thomas Tabor Nouvelles approches de vecteur génétique destinées à l'esthétique, la chirurgie plastique esthétique, la dermatologie cosmétique et d'autres utilisations
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