Novel roles of bovine milk‐derived exosomes in skin antiaging

Exosomes are small vesicles released from cells and are found in various mammalian biological fluids, such as bovine milk, which has been employed in skincare for many years, apart from its dairy applications. In addition, exosomes have been recognized as vehicles for intercellular communication.


| BACKG ROU N D
Bovine milk serves as a raw material in the food industry and finds extensive application in the cosmetic sector due to its considerable biological potential, primarily attributed to casein and whey proteins. 1,2Milk-based products demonstrate positive effects on skin conditions, including improved wound healing, increased elasticity, and improved moisturization when incorporated into topical creams, ointments, etc. [3][4][5] Skin, a protective barrier in all animals, including humans, undergoes specific changes in individuals with age, with the most conspicuous manifestations being wrinkles, dryness, and a loss of natural smoothness. 6,7While the process of skin aging is a complex biological process, the underlying mechanisms remain incompletely understood.][10][11] To combat these effects, several antiaging strategies have been developed, such as skincare regimens, moisturizing preparations, and anti-wrinkle treatments.
Wrinkles primarily result from the loss of skin's elasticity; therefore, inhibiting the degradation of elastic fibers plays a central role in anti-wrinkle treatments. 12Hyaluronic acid and sericin have been employed to maintain skin hydration and combat the formation of wrinkles. 13,14osomes (40-150 nm in diameter) constitute a specific subtype of extracellular vesicles that originate within the endocytotic compartment.They are released by nearly all types of cells through biological membrane fusion between multivesicular bodies and the cell membrane.6][17] These exosomes, once released, are ubiquitously present in intercellular space and various bodily fluids, including bovine milk. 18few studies have demonstrated the role of exosomes distributed in the skin in mediating skin conditions through intercellular crosstalk among various skin cell types.For example, Hu et al. reported that human dermal fibroblast-derived exosomes exhibit the ability to ameliorate skin photoaging. 19Liu et al. elucidated that exosomes derived from keratinocytes possess the capacity to regulate melanocyte pigmentation by transporting microRNA cargo. 20yond skin-derived exosomes, Kim et al. observed that exosomes derived from extraneous sources, such as milk, can suppress melanogenesis. 21vine milk is abundant in exosomes.While the use of bovine milk-derived ingredients in functional cosmetics is widely accepted, further research is needed to explore the effects of bovine milk-derived exosomes on skin conditions.Keratinocytes and fibroblasts, both constructional skin cell types, play essential roles in skin conditions, including moisturization and wrinkle reduction. 22Skin aging primarily results from alterations in the dermal extracellular matrix, leading to a significant decrease in Col I content.Additionally, approximately 80% of facial aging can be attributed to exposure to UV radiation from sunlight, which can also contribute to collagen degradation. 23In this study, we investigated the impact of MK-Exo on the functions of keratinocytes and fibroblasts, as well as changes in collagen content under UV exposure.
We also assessed the skin toxicity of MK-Exo in both animals and humans.Furthermore, we conducted validation studies to confirm the moisturizing and anti-wrinkling properties of MK-Exo in female volunteers.
Subsequently, we found that MK-Exo exhibit the capacity to elevate the expression levels of FLG and CD44 in keratinocytes, as well as HAS2 in fibroblasts.Moreover, they effectively mitigate the UV-induced reduction in collagen production.Additionally, MK-Exo significantly improve the migratory capabilities of fibroblasts.Subsequent to conducting skin toxicity assessments in both animal and human participants, we further substantiated the moisturizing and anti-wrinkling properties of MK-Exo in female volunteers.Our findings collectively underscore the safety and potential utility of MK-Exo as a novel ingredient in antiaging skincare formulations.

| Cell lines
CCC-ESF-1 and HaCaT cells were procured from the National Institute of Cell Resource, Beijing, China.CCC-ESF-1 cells were cultivated in DMEM (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 μg/mL streptomycin.HaCaT cells were cultured in MEM (Gibco) supplemented with 10% FBS and 100 μg/mL streptomycin.Both cell lines were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO 2 .

| Exosome preparation
Fresh bovine milk was obtained from a local dairy facility.Exosomes were isolated via density gradient centrifugation.Briefly, the pH of milk was adjusted to pH 4.6 using hydrochloric acid (Merck), followed by centrifugation at 4000 rpm (Beckman) at 4°C for 30 min.Subsequently, sucrose density gradient centrifugation was performed as described previously, 24 and the resulting MK-Exo were sterile-filtered through a 0.22-μm filter and stored at −80°C until further use.

| Quantitative assessment of protein concentration
The protein concentration of MK-Exo was determined using a BCA kit (Thermo, Waltham, MA, USA) following the manufacturer's instructions.

| Transmission electron microscopy
MK-Exo (100 μg/mL) were fixed with 2% (w/v) paraformaldehyde at room temperature for 15 min.Subsequently, the mixture (10 μL) was applied onto a formvar-carbon-coated grid (Beijing XXBR Technology) at room temperature for 3 min.The grid was then exposed to a uranyl oxalate solution (4% uranyl acetate, 0.0075 M oxalic acid; pH 7) for 1 min.Subsequently, the samples were examined by transmission electron microscopy (TEM) (Hitachi High-Technologies Corporation).

| Size distribution and quantity of MK-Exo
The size distribution and quantity of MK-Exo were determined using NanoFCM (NanoFCM Inc.) following the manufacturer's instructions.

| Purity analysis
Samples were analyzed using an SEC-1000 column (7.8 mm × 150 mm, 7 μm; Thermo).The column was eluted at a flow rate of 0.3 mL/min with a mobile phase consisting of 150 mM NaCl and 20 mM phosphate buffer at pH 7.2.

| Proteomic analysis
To elucidate the proteomic profile of MK-Exo, we conducted liquid chromatography/mass spectrometry (LC-MS/MS) analysis, as previously described with a few modifications. 26The LC-MS/MS analysis was performed utilizing Easy NLC 1200-Q Exactive Orbitrap Mass Spectrometers (Thermo).The nano-HPLC system featured an Acclaim PepMap nano-trap column (C18, 100 Å, 75 μm × 2 cm) for sample enrichment and an Acclaim Pepmap RSLC analytical column (C18, 100 Å, 75 μm × 25 cm) for separation.Typically, a volume of 1 μL of the peptide mixture was loaded onto the enrichment (trap) column.All mass spectra were acquired in positive mode using full-scan MS spectra scanning across the FT mode ranging from m/z 300 to 1650, with a resolution of 70 000.For MS/MS, we selected the 15 most intense ions with charge states ≥2, isolated them with an isolation window of 1.6 m/z, and subjected them to HCD with a normalized collision energy of 28.Additionally, we applied a dynamic exclusion period of 30 s.

| RNA isolation
According to the manufacturer's instructions, total RNA was isolated from cells using Trizol (Thermo).

| RT-qPCR
For RT-qPCR analysis, PrimeScript™ RT Master Mix (Takara) and SYBR Green Premix Pro Taq HS (Takara) were employed.The primer details can be found in Table S1.

| MK-Exo uptake analysis
A total of 2 × 10 4 cells/well were seeded onto chamber slides and incubated overnight.Subsequently, the cells were exposed to AIElabeled MK-Exo (10 μg/mL) for 4 h, following fixation with 4% paraformaldehyde for 20 min.Nuclei were stained with DAPI (Thermo) and observed using a confocal laser-scanning microscope.

| Cell migration assays
A wound-healing assay was employed to assess cell migration.
Coculture experiments were performed by seeding CCC-ESF-1 cells (2 × 10 4 cells/well) and culturing them with varying concentrations of MK-Exo for 24 h.The migration rate was subsequently quantified by automated measurement of wound width (CBM, Biotek).

| Repeated skin irritation test
Female New Zealand rabbits (n = 4) were topically treated with a 0.5 g sample (PBS, MK-Exo) on both sides of the spine for a duration of 14 days.Erythema and oedema were assessed and recorded 1 h after each application.

| Skin photo-irritation test
In the case of the Dunkin Hartley rabbits (males: n = 3, females: n = 3), the spine was divided into four distinct treatment sites (left 1, 3 and right 2, 4).Samples 1 and 2 were applied for 30 min, after which sites 1 and 3 were covered with tin foil, followed by UVA irradiation at a dosage of 1000 mJ/cm 2 .Subsequently, skin reactions were observed at various time points (1, 24, 48, and 72 h).

| Skin photoallergy test
Male Dunkin Hartley were randomly assigned to one of three groups: NC (PBS, n = 10), PC (2,4-methylcoumarin, n = 10), and MK-Exo (n = 10).The initial phase involved the administration of 0.1 mL of sensitizer (Freund's Adjuvant Complete mixed with normal saline in a 1:1 ratio) at four distinct points in the neck hair removal area.

| Skin patch test
The patch test equipment utilized had an area not exceeding 50 mm 2 and a depth of approximately 1 mm.A sample was then introduced into the patch tester with a dosage ranging from 0.02 mL to 0.025 mL, with the NC (PBS) and MK-Exo groups distinguished.The patch tester was affixed to the curved side of the subject's forearm for 24 h.Following the removal of the patch tester, observations of skin reactions were conducted at 0.5, 24, and 48 h.

| Skin moisturization and wrinkle test
A cohort of healthy Chinese female individuals (n = 31, age range: 26-45 years) participated in the study, with all volunteers providing signed informed consent forms.MK-Exo were diluted with water to a concentration of 60 μg/mL and applied morning and evening for a period of 28 days.The efficacy of MK-Exo was assessed using VISIA, measuring skin hydration (Corneometer CM825), skin elasticity (Cutometer MPA580), skin wrinkle count, and wrinkle area (PRIMOS CR).

| Statistical analyses
The specific statistical methods employed for each experiment are detailed in the corresponding figure legends.All experiments were conducted with a minimum of three replicates, and results were considered statistically significant at p < 0.05.

| Characterization and labelling of MK-Exo
To acquire MK-Exo samples for our study, we isolated them from fresh bovine milk (Figure 1A).We employed morphological analysis and detected specific protein markers (CD9 and TSG101) to identify the fractions containing MK-Exo (Figure 1B,C) following DC.Subsequently, we confirmed the presence of MK-Exo through size distribution analysis and the presence of exosomal positive and negative markers (Figure 1D,F).The high purity of MK-Exo was indicated by a distinct peak in the HPLC profile (Figure 1G).These results affirmed the successful isolation of highly pure MK-Exo for our research.In our quest to trace MK-Exo in subsequent analyses, we assessed the labeling efficiency of hydrophobic fluorescent dyes commonly used for exosome tracing, both in vitro and in vivo.
Our observations revealed substantial variation in the labeling

| Effects of MK-Exo on human keratinocytes
To investigate the potential impact of MK-Exo on the moisturizing functions of keratinocytes, we first analyzed MK-Exo using HaCaT cells.We observed the cellular uptake of MK-Exo (Figure 2A).These preliminary findings suggest that MK-Exo may exert its effects on human keratinocytes, transcending species boundaries.
To further investigate whether MK-Exo can induce changes in gene expression associated with moisture regulation in keratinocytes in vitro, we explored the mRNA and protein expression of select genes recognized as indicators of moisturization.We found that MK-Exo had the capacity to enhance the expression of FLG, a natural moisturizing factor, by approximately threefold (Figure 2B,C), as well as augment the expression of CD44, the receptor for hyaluronan acid, by more than 60% (Figure 2B,E).However, we found that at relatively high concentrations, MK-Exo led to a reduction in the levels of AQP3, the most abundant skin aquaporin (Figure 2B,D).

These results collectively indicate that MK-Exo may contribute
to moisture regulation by upregulating the expression of FLG and CD44 in keratinocytes without a change in AQP3 expression.

| Effects of MK-Exo on human fibroblasts
To determine the potential impact of MK-Exo on fibroblast functionality, we initially analyzed MK-Exo using CCC-ESF-1 cells.We found that these cells exhibited an ability to internalize MK-Exo (Figure 3A).This preliminary finding hints at the possibility of MK-Exo exerting its effects across species, specifically on human skin fibroblasts.
To further investigate whether MK-Exo has the capacity to influence the moisturizing function of human fibroblasts in vitro, we examined the protein levels of HAS2.We found that MK-Exo could elevate the expression level of HAS2 by more than twofold (Figure 3B).Elevated HAS2 levels correspond to enhanced synthesis of hyaluronan acid.These results suggest that MK-Exo may additionally play a role in moisturization by inducing the production of hyaluronan acid, a natural moisture factor, through HAS2.

| Primary safety evaluation of MK-Exo in skin
As the potential use of MK-Exo as a cosmetic material is considered, skin contact emerges as one of the primary exposure routes during routine daily use.Nevertheless, the toxic effects of MK-Exo exposure on animal and human skin through cutaneous contact have remained largely unexplored.Therefore, our initial objective was to assess the potential toxicity of MK-Exo on the skin of animals.This assessment involved skin allergy, skin photoallergy, repeated skin irritation, and skin photo-irritation tests.In the skin allergy, skin photoallergy (Table 1), and repeated skin irritation tests (Table S2), no allergic reactions were observed subsequent to sensitization with MK-Exo.Furthermore, the skin photo-irritation tests did not reveal any evidence of skin damage (Table S3).
To further investigate the potential for sensitization on human skin, we recruited 31 female volunteers for a patch test.The results of this test, conducted at intervals of 0.5, 24, and 48 h, indicated an absence of any skin reactions in all participants (Table S4).
In conclusion, these findings collectively indicate the lack of sensitization and irritant potential of MK-Exo in both animal and human skin.Consequently, there appears to be no risk associated with conducting efficacy evaluations of MK-Exo in humans.
The proportion of animals exhibiting a response to the number of tested animals in the erythema and oedema column is the skin reaction score (0, 1, 2, 3, or 4).The sensitization rate is the percentage of the number of animals with a response score of 2 or greater by the total number of animals in the respective group.

| Antiaging effect of MK-Exo in humans
We enrolled 31 female volunteers, aged between 26 and 45 years.
Following patch tests, all participants applied MK-Exo to their facial skin twice daily for 28 days, refraining from using other cosmetic products during this period.Skin conditions were assessed on days 2, 14, and 28.The moisture content of the skin exhibited an increase of 4.64% on Day 14 and 5.6% on Day 28 across all age groups.
Interestingly, the rise in moisture content was more pronounced in  2).
Furthermore, there was a dramatic improvement in skin brightness observed on Day 28.More than 90% of the participants reported satisfaction with the moisturizing effects of MK-Exo (data not shown).In addition, facial skin gloss, as depicted in photographs, displayed an enhancement on Day 28 (data not shown).The results collectively indicated that MK-Exo possesses moisturizing properties.Skin elasticity was observed on both Day 14 and Day 28.We observed increases in the F3/F4 value and R2 value by 6.33% and 7.24%, respectively, by Day 28.Furthermore, a substantial 10.74% increment was observed in the participants aged 36-45 years (Table 2).In addition, assessments of wrinkle area and the quantity of wrinkles revealed reductions of 9.37% and 5.27% on Day 14 and 9.59% and 4.99% on Day 28, respectively (Table 2).These results suggest a potential anti-wrinkle effect of MK-Exo in humans.

| DISCUSS ION
This study represents the first comprehensive evaluation of the safety and efficacy of MK-Exo on both animal and human skin.
Furthermore, it provides the initial evidence supporting the potential of MK-Exo in antiaging skincare, specifically through its moisturizing and anti-wrinkle properties in humans.Additionally, our research revealed a novel aspect of MK-Exo's functionality, demonstrating its capacity to modulate the expression of genes such as FLG, CD44, HAS2, and collagen genes.
The primary mechanism underlying MK-Exo's effects appears to involve direct interactions with cells.Our findings indicate that MK-Exo may exert its effects on sepsis, at least partially, through internalization by the two principal types of constitutional cells found in human skin, as observed in in vitro experiments.However, this finding may be confirmed by exploring whether inhibiting the uptake of MK-Exo in vitro can preserve its functional properties.When applied to human skin, MK-Exo encounters its first physical barrier in the form of the stratum corneum.However, the precise mechanism by which MK-Exo traverses this barrier remains unknown and necessitates further exploration in future studies.Skin aging primarily results from alterations in the dermal extracellular matrix and vice versa.In general, FLG undergoes degradation into free amino acids and amino acid derivatives within the intercellular space.This degradation process contributes to the hydration of the stratum corneum and enhances its UVB absorption capacity.FLG is a key component of the natural moisturizing factors.Increased FLG production within keratinocytes correlates with improved skin condition through enhanced natural moisturization.Elevated expression levels of HAS2, an enzyme responsible for hyaluronic acid synthesis, in fibroblasts and increased CD44 expression, a receptor that binds to hyaluronic acid on the outer cell membrane of keratinocytes, result in heightened hyaluronic acid content.
The interaction between HAS2 and CD44 facilitates the retention of hyaluronic acid within the skin.In summary, as previously discussed, MK-Exo can effectively function as a complex moisturizing agent.
Significantly reducing Col I content in the skin is a prominent factor contributing to the aging process.Approximately 80% of facial aging can be attributed to exposure to UV radiation from sunlight, which can also lead to collagen degradation. 23Our findings indicate that MK-Exo have the potential to mitigate this process and play a role in antiaging interventions.
Our study corroborates that MK-Exo can facilitate the migration of fibroblasts.The migration and proliferation of fibroblasts are essential components of the wound-healing process.The MK-Exo tests conducted on volunteers spanned only 28 days, during which its effect progressively intensified and may potentially continue to do so over an extended period.Interestingly, we found the MK-Exo exhibit divergent functionality across different age cohorts in specific test parameters.MK-Exo may exert a more pronounced effect on skin antiaging among older females.Indeed, more participants may be necessary for validation.
However, the findings may suggest that distinct age cohorts manifest unique skin conditions and respond differently to MK-Exo.Therefore, the underlying mechanism should be explored comprehensively.
Nevertheless, the precise mechanism of MK-Exo and the identification of its critical molecular components remain elusive.MK-Exo comprises a diverse array of proteins and noncoding RNAs, numbering in the thousands.Omics technology can be employed to identify the key elements, [27][28][29] that is, the specific proteins or RNAs responsible for instigating alterations in various skin biological processes.Through the application of GO enrichment analysis, one can scrutinize the assortment of proteins and mRNA molecules within MK-Exo that are associated with cellular senescence, cellular oxidative detoxification, and tight junctions.This analysis aims to pinpoint the critical molecules through which MK-Exo exert their antiaging effects.Furthermore, differential expression profiling of proteins in fibroblasts and keratinocytes subsequent to MK-Exo uptake can serve as a means to substantiate the antiaging efficacy of MK-Exo in subsequent investigations.These key molecular components will serve as essential quality attributes and will occupy a central role in the quality control framework of the industrial process.This study has the potential to provide more robust evidence supporting the viability of MK-Exo as a cosmetic material.
MK-Exo demonstrated significant potential for enhancing skin moisturisation and antiaging effects.However, a critical challenge in utilizing them as a novel cosmetic ingredient lies in preserving their activity throughout preparation and storage.At present, The raw files were analyzed using Proteome Discover (version 2.4; Thermo) with Sequest employed as the search engine.Fragment and peptide mass tolerances were defined at 20 mDa and 10 ppm, respectively, permitting a maximum of two missed cleavage sites.The false discovery rates for both proteins and peptides were set at 1%.To determine the most significantly enriched signal transduction pathways within the dataset, we utilized the DAVID Bioinformatics Resource 2021 (http:// david.abcc.ncifc rf.gov/ ), applying recommended analytical parameters to analyze the differential expression of proteins.

| 1379 LU
et al. efficiency among different dyes, ranging from 15.7% to over 90% (Figure S1A,D).The dye known as AIE demonstrated the highest labeling efficiency.Therefore, for precision in exosome tracing, we labeled MK-Exo with AIE in our subsequent investigations.
Furthermore, we examined alterations in collagen content following UV exposure to investigate whether MK-Exo could affect wrinkle formation in relation to collagen status.The results showed that MK-Exo did not significantly change the expression levels of Col I and Col III.Instead, it appeared to mitigate the reductions in Col I and Col III observed in fibroblasts following UV exposure (Figure 3C,E).In addition, we observed that MK-Exo could enhance fibroblast cell migration (Figure 3F, Figure S2) rather than keratinocytes (data not shown).These results suggest that MK-Exo may function as an anti-wrinkling agent by attenuating collagen degradation and enhancing cell migration.

F I G U R E 1
Preparation and characterization of MK-Exo.(A) Flow chart of the preparation of MK-Exo via density gradient ultracentrifugation.Each fraction of density gradient ultracentrifugation was analyzed by western blot of CD9 and TSG101 (B), and the morphological characteristics of MK-Exo were identified by TEM (C).(D) The F3 and F4 fractions were mixed, and particle size was determined by NanoFCM.(E) LC-MS/MS was performed to analyze positive and negative markers of exosomes.(F) The expression of CD81, TSG101, GM130, and calnexin was analyzed by western blot.(G) HPLC was performed to determine the purity of MK-Exo.| 1381 LU et al. volunteers aged 36-45 years, whereas the increase among those aged 26-35 years did not achieve statistical significance (Table

F I G U R E 2 2
Effect of MK-Exo on HaCaT cells.(A) Representative fluorescence images of AIE-labeled MK-Exo uptake by HaCaT cells.(B) The protein expression levels of FLG, APQ3, and CD44 were determined by western blot.The transcript levels of FLG (C), APQ3 (D), and CD44 (E) genes as determined by RT-qPCR.An asterisk (*) represents p < 0.05; double asterisks (**) represent p < 0.01; and triple asterisks (***) represent p < 0.001.| 1383 LU et al. conventional exosome storage methods involve low temperatures (−80°C).Unfortunately, this approach not only escalates the expenses associated with raw material production and transportation but also restricts the maintenance of exosome activity without the use of protective agents, limiting their applicability in cosmetic formulations.Freeze-drying is a technique employed to convert substances into lyophilized powder.Numerous active biological and cosmetic raw materials have been successfully freeze-dried, allowing for their long-term storage at temperatures ranging from 2°C to 8°C while retaining their efficacy.Therefore, the adoption of freeze-drying technology can enable the transformation of MK-Exo into lyophilized powder, facilitating their TA B L E 1 Skin allergy and skin photoallergy tests.Skin moisturization and wrinkle tests.

F I G U R E 3
Effect of MK-Exo on CCC-ESF-1 cells.(A) Representative fluorescence images of AIE-labeled MK-Exo uptake by CCC-ESF-1 cells.(B) The protein expression levels of HAS2 and GAPDH were analyzed by western blot.The expression of Col I and Col III was determined by western blot (C) and RT-qPCR (D, E). (F) The variation in scratch width at different time points.An asterisk (*) represents p < 0.05, and double asterisks (**) indicate p < 0.01.