Application of mesenchymal stem cell exosome subdivision subgroup S2

Abstract

The invention relates to the technical field of biology, and provides an application of a mesenchymal stem cell exosome subgroup S2 in preparation of a cosmetic composition or a medicine for improving skin. The cosmetic composition or medicine takes the mesenchymal stem cell exosome subgroup S2 as a single active ingredient. Compared with non-subdivided exosomes, the exosome has the advantages of definite function and stronger effect, and can realize more efficient and more stable skin improvement effect through the unique biological characteristics of the exosome.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of biomedicine and dermatology, and specifically relates to the use of a subpopulation S2 of mesenchymal stem cell exosomes in the preparation of skin-improving cosmetics or medicines. Background Art

[0002] Exosomes are nanoscale vesicles (30-150 nm) secreted by cells. They carry bioactive molecules such as proteins, nucleic acids, and lipids, and have intercellular communication and regulatory functions. Mesenchymal stem cells (MSCs) are adult multipotent stem cells with the ability to differentiate, proliferate, and self-renew. Exosomes derived from MSCs offer several advantages over MSCs in terms of efficacy and clinical applications.

[0003] However, stem cell exosomes still have related limitations: 1) Heterogeneity: Exosomes extracted by traditional methods are mixed populations, which makes them prone to instability between batches and leads to unstable efficacy; 2) Limited effect: Unoptimized and subdivided exosome mixtures are not efficient in promoting collagen synthesis, inhibiting melanin production, etc., and are difficult to meet clinical or cosmetic needs.

[0004] It is clear that the heterogeneity of exosomes and other characteristics have hindered their clinical application. These problems are ultimately related to the lack of research on the specific functions and molecular components of exosome subpopulations. Therefore, the effective application of exosome drugs or products is based on the careful and in-depth study of specific exosome subpopulations with different characteristics and functions, and the effective verification and development of the functional effects of exosome subpopulations. Summary of the Invention

[0005] The present invention provides an exosome subpopulation S2 with clear functions and stronger effects. Through its unique biological characteristics, it solves the above-mentioned technical problems and achieves a more efficient and stable skin improvement effect.

[0006] The present invention provides a use of a mesenchymal stem cell exosome subpopulation S2 in preparing a cosmetic composition or a medicine for improving skin. The cosmetic composition or medicine uses the mesenchymal stem cell exosome subpopulation S2 as an active ingredient. The mesenchymal stem cell exosome subpopulation S2 has a peak particle size range of 90 nm ± 15 nm and an average particle size of 100 ± 15 nm, and surface markers include B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

[0007] As an alternative, the surface markers of mesenchymal stem cell exosome subpopulation S2 were high expression of CD63, low expression of CD9, and negative expression of ALIX, and the exosome purity was 1.01×108 particles / μg protein.

[0008] As an optional option, the method for preparing the mesenchymal stem cell exosome subpopulation S2 includes: isolating and culturing human umbilical cord-derived mesenchymal stem cells, collecting the culture medium supernatant; filtering; using a tangential flow filtration system to obtain a concentrated solution from the filtered supernatant, and separating and purifying the concentrated solution by size exclusion chromatography and / or affinity chromatography to obtain the exosome subpopulation S2.

[0009] As an alternative, when the concentrate is separated and purified by size exclusion chromatography, the component corresponding to the second UV absorption peak in the eluate is collected as the exosome subpopulation S2.

[0010] As an optional option, the preparation method includes: selecting and isolating umbilical cord-derived mesenchymal stem cells obtained from healthy pregnant women within the seventh generation and in the logarithmic growth phase; inoculating the mesenchymal stem cells in DMEM / F12 culture medium containing 10% fetal bovine serum, and culturing them to the fifth to seventh generations until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, and then replacing them with serum-free culture medium, continuing to culture, and collecting the cell culture supernatant containing exosomes.

[0011] As an optional option, the filtration, concentration, and separation and purification steps in the preparation method include: deep filtering the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment liquid; concentrating the pretreatment liquid through a tangential flow ultrafiltration system, and then separating and purifying it by size exclusion chromatography, collecting the component corresponding to the second ultraviolet absorption peak in the eluate to obtain the exosome subpopulation S2.

[0012] As an alternative, the concentration of the exosome subpopulation S2 in the cosmetic composition or medicament is 1×10 8 ~1×10¹¹ particles / mL.

[0013] Alternatively, the cosmetic composition or drug is in the form of a lyophilized powder, a hydrogel or a sterile solution.

[0014] As an optional solution, the use for skin improvement includes: treating or preventing photoaging or skin barrier damage, promoting skin elasticity recovery, whitening, wrinkle removal, moisturizing, and reducing or eliminating post-inflammatory pigmentation.

[0015] As an optional solution, the skin improvement includes at least one of the following situations: a) Promote fibroblast proliferation and / or collagen and elastin synthesis; b) Promote keratinocyte proliferation; c) Promote the proliferation of vascular endothelial cells; d) inhibiting macrophage inflammatory response; e) Inhibit melanin synthesis in melanoma cells; f) Scavenging oxygen free radicals.

[0016] This study reveals for the first time the synergistic effects of exosome subpopulation S2 as a single active ingredient in promoting skin cell proliferation, inhibiting inflammation and melanin production, and scavenging oxygen free radicals. This discovery could be used to develop functional cosmetics or pharmaceuticals for the treatment of photoaging, skin barrier damage, and pigmentary abnormalities. Compared to unsubpopulated exosomes, the subpopulation S2 of this invention significantly promotes fibroblast proliferation, enhances collagen and elastin synthesis, significantly promotes keratinocyte proliferation, significantly scavenges oxygen free radicals, significantly inhibits melanin synthesis in melanoma cells, significantly promotes endothelial cell proliferation, and effectively inhibits macrophage inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effects of exosome subpopulation S2 and unsubpopulated exosomes on fibroblast proliferation, collagen and elastin synthesis were analyzed.

[0018] Figure 2 The effects of exosome subpopulation S2 and unsubpopulated exosomes on keratinocyte proliferation.

[0019] Figure 3 The effects of exosome subpopulation S2 and unsubpopulated exosomes exo in scavenging oxygen free radicals.

[0020] Figure 4 The effects of exosome subpopulation S2 and unsubpopulated exosomes exo on melanin synthesis in melanoma cells.

[0021] Figure 5 The effects of exosome subpopulation S2 and unsubpopulated exosomes exo on vascular endothelial cell proliferation.

[0022] Figure 6 The effects of exosome subpopulation S2 and unsubpopulated exosomes on the inflammatory response of macrophages. DETAILED DESCRIPTION

[0023] The present invention provides a new medical use of the mesenchymal stem cell-derived exosome subpopulation S2, which, as an active ingredient, has been confirmed for the first time by the present invention to be useful in preparing skin-improving products, such as cosmetic compositions or medicines.

[0024] Exosomes derived from mesenchymal stem cells (MSCs) possess anti-inflammatory, pro-regenerative, and immunomodulatory properties, and can be used for skin repair, anti-aging, and disease treatment. They can promote wound healing, stimulate collagen synthesis, slow skin aging, and repair skin problems such as inflammation and redness, thus holding great promise for application in the medical aesthetics field. However, due to heterogeneity and other factors, exosome mixtures without specific subpopulations are limited in their effectiveness when applied to human skin as cosmetics or pharmaceuticals, making their translation into highly active products difficult.

[0025] By subdividing mesenchymal stem cell exosomes into subpopulations, the research team discovered and validated the significant efficacy of this exosome subpopulation in improving skin quality while improving the homogeneity of the exosome product. The high purity of these subpopulations allows for scalable production and the development of superior cosmetic or pharmaceutical products, which holds significant implications for the application of exosomes in medical aesthetics.

[0026] Specifically, the mesenchymal stem cell-derived exosome subpopulation S2 of the present invention is defined and limited by the following characteristics or the following preparation method.

[0027] The particle size of the mesenchymal stem cell exosome subpopulation S2 has a peak value of 90nm±15nm and an average particle size of 100±15nm. Surface markers include B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN. More specifically, in some embodiments, the surface markers of the mesenchymal stem cell exosome subpopulation S2 are high expression of CD63, low expression of CD9, and negative expression of ALIX. The exosome purity is 1.01×10 8 particles / μg protein.

[0028] The method for preparing the mesenchymal stem cell exosome subpopulation S2 comprises: isolating and culturing human umbilical cord-derived mesenchymal stem cells, collecting the culture supernatant thereof; filtering; subjecting the filtered supernatant to a tangential flow filtration system to obtain a concentrate, and separating and purifying the concentrate by size exclusion chromatography and / or reverse affinity chromatography to obtain the exosome subpopulation S2.

[0029] In some embodiments, the method for preparing the mesenchymal stem cell exosome subpopulation S2 comprises the following steps: 1) Cell culture supernatant collection: Umbilical cord mesenchymal stem cells were cultured to passages 5-7 and the cell culture supernatant within this passage range was collected; 2) Pretreatment: The supernatant was filtered to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; 3) Exosome subpopulation separation: The pretreatment solution is concentrated by a tangential flow ultrafiltration system and then separated and purified by size exclusion chromatography. The components corresponding to the second ultraviolet absorption peak in the eluate are collected to obtain the target exosome subpopulation S2.

[0030] In some more specific schemes, the preparation method includes: selecting and isolating umbilical cord-derived mesenchymal stem cells obtained from healthy pregnant women within the seventh generation and in the logarithmic growth phase; inoculating the mesenchymal stem cells in DMEM / F12 medium containing 10% fetal bovine serum, and subculturing them to the fifth to seventh generations until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, and then replacing them with serum-free medium, continuing to culture, and collecting the cell culture supernatant containing exosomes; deep filtering the cell culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentrating the pretreatment solution through a tangential flow ultrafiltration system, separating and purifying it through size exclusion chromatography, and collecting the component corresponding to the second ultraviolet absorption peak in the eluate to obtain the exosome subpopulation S2.

[0031] In addition, in some schemes, the preparation method and operation details of the above-mentioned exosome subpopulation S2 are the same or substantially the same as the relevant scheme in patent application text CN118222495A.

[0032] The exosome subpopulation S2 is the only active ingredient of the cosmetic composition or medicine of the present invention. In a preferred embodiment, it can be made into a lyophilized powder, a hydrogel or an injection, and the concentration is 1×10 8 ~1×10¹¹ particles / mL, suitable for the treatment of photoaging, inflammatory skin diseases and pigmentation.

[0033] The use for skin improvement described in the present invention specifically includes: treating or preventing photoaging or skin barrier damage, promoting skin elasticity recovery, whitening, wrinkle removal, moisturizing, and reducing or eliminating post-inflammatory pigmentation.

[0034] The skin improvement includes at least one of the following situations or achieves at least one of the following effects: a) Promote fibroblast proliferation and / or collagen and elastin synthesis; b) Promote keratinocyte proliferation; c) Promote the proliferation of vascular endothelial cells; d) inhibiting macrophage inflammatory response; e) Inhibit melanin synthesis in melanoma cells; f) Scavenging oxygen free radicals.

[0035] In some embodiments, the exosome subpopulation S2 of the present invention is significantly better than the exosomes of the unsubpopulated subpopulation in the above-mentioned effects or is at least equivalent to the exosomes of the unsubpopulated subpopulation.

[0036] Example 1 Preparation and Identification of Mesenchymal Stem Cell Exosome Subpopulation S2 (1) Preparation of mesenchymal stem cell culture supernatant and pretreatment solution: Umbilical cord mesenchymal stem cells were cultured in a CO2 incubator to P5-7 generations with cell viability greater than 80%. The cell culture solution was deep filtered to remove large vesicles and apoptotic bodies to obtain mesenchymal stem cell culture pretreatment solution.

[0037] (2) Concentration of mesenchymal stem cell culture supernatant: The mesenchymal stem cell culture supernatant was concentrated 20 times to obtain a concentrate using a tangential flow filtration concentration system. The tangential flow concentration system used a 300 kDa hollow fiber filter, PBS was washed 16 to 25 times, the transmembrane pressure (TMP) was 0.5 bar, and the flow rate was 385 LMH.

[0038] (3) Size Exclusion Chromatography: The above product was further purified by the following steps: Equilibration: Equilibrate a Sephacryl S-400 HR gel filtration column with PBS buffer for 2 CV.

[0039] Sample loading: Sample loading capacity 0.5~30mg / mL.

[0040] Elution: Wash with PBS buffer, start collecting when the UV signal rises, stop collecting when it approaches the baseline level, and collect the second UV absorption peak to subdivide the exosome subpopulation S2.

[0041] Cleaning in place (CIP): Rinse with 0.5M NaOH solution for 2CV.

[0042] (4) Exosome identification: Transmission electron microscopy (TEM) observations showed that the exosome subpopulation S2 had a classic saucer-like morphology; nanoparticle tracking analysis (NTA) showed that the S2 particle size range peaked at 90 nm ± 15 nm and the average particle size was 100 ± 15 nm. Western blotting experiments showed that S2 surface markers included B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

[0043] The following Examples 2-7 studied and verified the function and activity of the exosome subpopulation S2. The exosome subpopulation S2 used was the exosome subpopulation S2 prepared in Example 1.

[0044] Example 2 Fibroblast proliferation, collagen and elastin synthesis experiments The cell proliferation ability was detected by CCK8 method: HSF in the logarithmic growth phase was cultured at 4×10 4 / mL cell density was inoculated into 96-well cell culture plates, and the culture supernatant was discarded after the cells adhered to the wall and replaced with DMEM / F12 basal medium. The experimental group was supplemented with 2×10 9 (L) and 1×10 10 (H) Undifferentiated exosomes (exo) and subdifferentiated exosomes (S2) were analyzed. The negative control group received an equal volume of PBS. Six replicate wells were set up in each group. After 72 hours of culture, the cell culture supernatant was collected and centrifuged at 2000 × g for 5 minutes. The supernatant was then used for enzyme-linked immunosorbent assay (ELISA) to detect collagen and elastin expression. 100 μl of DMEM / F12 basal medium and 10 μl of CCK8 solution were added to each well of the cell culture plate and incubated for 30 minutes. The absorbance was measured at 450 nm (A). Proliferation promotion (%) = OD experimental group / OD blank group × 100%.

[0045] from Figure 1 The results show that in terms of cell proliferation indicators, the effects of high and low concentrations of the exosome subpopulation S2 are significantly better than those of the unsubdivided exosome subpopulation exo ( Figure 1 A) For collagen index, although there is no significant difference between S2 and exo at high concentrations, S2 is significantly better than exo at low concentrations ( Figure 1 B), while elastin showed a significant difference at low concentrations ( Figure 1 C) In general, S2 is better than exo.

[0046] Example 3 Keratinocyte proliferation experiment The cell proliferation ability was detected by CCK8 method: HaCaT cells in the logarithmic growth phase were cultured at 3×10 4 / mL cell density was inoculated into 96-well cell culture plates, and the culture supernatant was discarded after the cells adhered to the wall and replaced with DMEM basal medium. The experimental group was supplemented with 2×10 9 (L) and 1×10 10 (H) For the undifferentiated exosome subpopulations exo and the subdifferentiated exosome subpopulation S2, an equal amount of PBS was added to the blank group. Six replicate wells were set up in each group. After 72 h of culture, the old culture medium was discarded, and 100 μl of DMEM basal culture medium and 10 μl of CCK8 solution were added to each well and incubated for another 30 min. The absorbance was measured at 450 nm (A). The proliferation promotion rate (%) = OD experimental group / OD blank group × 100%.

[0047] from Figure 2 The results show that the exo group has a weaker effect and only has a significant promoting effect at high concentrations, while S2 can significantly promote keratinocyte proliferation at both high and low concentrations, indicating that S2 is more effective than exo.

[0048] Example 4 ABTS free radical scavenging experiment Mix equal volumes of ABTS solution and oxidant solution to form ABTS working stock solution, store in the dark at room temperature for 12-16 hours before use, and dilute 50 times with PBS to form ABTS working solution before use. Add 200 μl of ABTS working solution to each well of a 96-well cell culture plate. Add 2×10 9 (L) and 1×10 10 (H) For the undifferentiated exosomes and subpopulation S2, an equal amount of PBS was added to the blank group. Three replicate wells were set up for each group. After incubation at room temperature for 2-6 minutes, the absorbance was measured at 734 nm (A). The clearance efficiency (%) = (OD blank group - OD experimental group) / OD blank group × 100%.

[0049] from Figure 3 The results showed that both S2 and exo could significantly scavenge ABTS free radicals in a dose-dependent manner, but the scavenging effect of S2 was significantly better than that of exo.

[0050] Example 5 Melanoma cell melanin synthesis experiment B16-F10 in the logarithmic growth phase was grown at 8×10 4 / mL cell density was inoculated into 6-well cell culture plates, and the culture supernatant was discarded after the cells adhered to the wall and replaced with DMEM complete medium containing 10% FBS. The experimental group was supplemented with 2×10 9 (L) and 1×10 10 (H) Undifferentiated exosomes and subdivided S2. The blank group was added with an equal amount of PBS. Each group was also added with 0.2 μM α-MSH. Each group had three replicate wells. After 72 h of culture, the cell culture supernatant was collected and centrifuged at 2000 × g for 5 minutes. The supernatant was then used to detect melanin expression by enzyme-linked immunosorbent assay.

[0051] from Figure 4 The results show that both S2 and exo can significantly inhibit melanin synthesis in a dose-dependent manner. Although the inhibitory effect of S2 is comparable to that of exo at low concentrations, the effect is significantly better than exo at high concentrations. In general, S2 is better than exo.

[0052] Example 6 Vascular endothelial cell proliferation experiment The cell proliferation ability was detected by CCK8 method: bEnd.3 cells in the logarithmic growth phase were cultured at 3×10 4 / mL cell density was inoculated into 96-well cell culture plates, and the culture supernatant was discarded after the cells adhered to the wall and replaced with DMEM basal medium. The experimental group was supplemented with 2×10 9 (L) and 1×10 10(H) For the undifferentiated exosomes and subpopulation S2, an equal amount of PBS was added to the blank group. Six replicate wells were set up for each group and cultured for 72 h. The old culture medium was discarded and 100 μl of DMEM basal medium and 10 μl of CCK8 solution were added to each well for a further 30 min. The absorbance was measured at 450 nm (A). The proliferation promotion rate (%) = OD experimental group / OD blank group × 100%.

[0053] from Figure 5 The results showed that both S2 and exo could significantly promote the proliferation of vascular endothelial cells, but the effect of S2 was significantly better than that of exo.

[0054] Example 7 Macrophage Inflammation Inhibition Experiment RAW264.7 in the logarithmic growth phase was used at 2×10 5 / mL cell density was inoculated into 6-well cell culture plates, and the culture supernatant was discarded after the cells adhered to the wall and replaced with DMEM complete medium containing 10% FBS. The experimental group was supplemented with 2×10 9 (L) and 1×10 10 (H) Undifferentiated exosomes (exo) and subdivided exosomes (S2). The blank group was treated with an equal amount of PBS. 100 ng / mL LPS was added to each group. Three replicates were set up for each group. After 24 h of culture, the cell culture supernatant was collected and centrifuged at 2000 × g for 5 minutes. The supernatant was then used to detect IL-6 expression by enzyme-linked immunosorbent assay.

[0055] from Figure 6 The results show that the inflammatory regulation ability of S2 is slightly weaker than that of exo, which may be because S2 is mainly responsible for cell regeneration and its inflammatory regulation ability is slightly weaker, but there is no significant difference with exo, indicating that S2 still has certain inflammatory regulation activity.

[0056] In summary, the exosome subpopulation S2 of the present invention has better activity in the field of skin improvement than the unsubdivided subpopulation exo, and has greater potential for future market applications.

[0057] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. Use of a mesenchymal stem cell exosome subpopulation S2 in the preparation of a cosmetic composition or drug for skin improvement, wherein the cosmetic composition or drug uses the mesenchymal stem cell exosome subpopulation S2 as an active ingredient; the mesenchymal stem cell exosome subpopulation S2 has a peak particle size range of 75nm to 105nm, an average particle size of 85nm to 115nm, and surface markers including B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

2. The use according to claim 1, characterized in that The surface markers of mesenchymal stem cell exosome subpopulation S2 showed high expression of CD63, low expression of CD9, and negative expression of ALIX. The purity of exosomes was 1.01×10 8 particles / μgprotein.

3. The use according to claim 1, characterized in that The preparation method of mesenchymal stem cell exosome subpopulation S2 includes: isolating and culturing human umbilical cord-derived mesenchymal stem cells, collecting their culture supernatant; filtering; using a tangential flow filtration system to obtain a concentrated solution from the filtered supernatant, and separating and purifying the concentrated solution by size exclusion chromatography and / or reverse affinity chromatography to obtain exosome subpopulation S2.

4. The use according to claim 3, characterized in that When the concentrate is separated and purified by size exclusion chromatography, the component corresponding to the second ultraviolet absorption peak in the eluate is collected as the exosome subpopulation S2.

5. The use according to claim 3, characterized in that The method for preparing the culture supernatant includes: selecting and isolating umbilical cord-derived mesenchymal stem cells obtained from healthy pregnant women within the seventh generation and in the logarithmic growth phase; inoculating the mesenchymal stem cells in DMEM / F12 culture medium containing 10% fetal bovine serum, culturing them, and subculturing them to the fifth to seventh generations until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, and then replacing them with serum-free culture medium, continuing to culture, and collecting the cell culture supernatant containing exosomes.

6. The use according to claim 3 or 5, characterized in that The filtration, concentration, and separation and purification steps in the preparation method include: deep filtering the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment liquid; concentrating the pretreatment liquid through a tangential flow ultrafiltration system, and then separating and purifying it by size exclusion chromatography, collecting the component corresponding to the second ultraviolet absorption peak in the eluate to obtain the exosome subpopulation S2.

7. The use according to claim 1, characterized in that The concentration of the exosome subpopulation S2 in the cosmetic composition or medicine is 1×10 8 ~1×10¹¹ particles / mL.

8. The use according to claim 1, characterized in that The dosage form of the cosmetic composition or medicine is freeze-dried powder, hydrogel or sterile solution.

9. The use according to claim 1, characterized in that The use for skin improvement includes: treating or preventing photoaging or skin barrier damage, promoting skin elasticity recovery, whitening, wrinkle removal, moisturizing, and reducing or eliminating post-inflammatory pigmentation.

10. The use according to claim 1, characterized in that The skin improvement includes at least one of the following situations: a) Promote fibroblast proliferation and / or collagen and elastin synthesis; b) Promote keratinocyte proliferation; c) Promote the proliferation of vascular endothelial cells; d) inhibiting macrophage inflammatory response; e) Inhibit melanin synthesis in melanoma cells; f) Scavenging oxygen free radicals.

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