1 Basic Concepts of Exosomes and Extracellular Vesicles

1.1 Exosomes and Extracellular Vesicles

Extracellular vesicles (EVs) are particles released by cells into the extracellular environment. They are enclosed by a lipid bilayer and are incapable of self-replication. Their lumens and membrane surfaces may carry proteins, lipids, messenger RNA (mRNA), microRNA (miRNA), metabolites, and other components.

Exosomes are a class of extracellular vesicles defined by their biogenesis and originate from the endosomal system. Their formation can be summarized as follows:

Plasma membrane endocytosis → early endosome → inward budding of the late endosomal membrane → formation of intraluminal vesicles → formation of multivesicular bodies → fusion of multivesicular bodies with the plasma membrane → release of intraluminal vesicles as exosomes

Multivesicular bodies may also fuse with lysosomes and undergo degradation:

Multivesicular body → fusion with the plasma membrane and release of exosomes

Multivesicular body → fusion with lysosomes and degradation

The endosomal sorting complex required for transport (ESCRT), tetraspanins, ceramides, and Rab-family small GTPases may participate in vesicle formation, cargo sorting, intracellular trafficking, and release. However, different cell types may use different molecular pathways.

The term “exosome” cannot be confirmed solely on the basis of particle size, cup-shaped morphology, or the presence of proteins such as CD9, CD63, and CD81. Small extracellular vesicles generally refer to vesicle populations with an isolated particle size of less than approximately 200 nm. Such populations may contain both endosome-derived exosomes and small vesicles formed by direct outward budding from the plasma membrane. Therefore, when the biogenetic origin has not been established, “extracellular vesicles” or “small extracellular vesicles” is a more accurate designation than the general term “exosomes.”

1.2 “Exosome Ingredients” on the Market May Represent Different Types of Materials

Ingredients described as exosomes in skincare products do not necessarily share the same material basis. Common categories include:

① Isolated and purified extracellular vesicles: These have undergone concentration and purification, with free proteins, culture-medium components, and non-vesicular particles removed to some extent.

② Extracellular vesicle-enriched fractions: These contain a higher vesicle concentration than the original conditioned medium but may still contain substantial amounts of free proteins, lipoproteins, nucleic acids, and residual culture-medium components.

③ Cell-conditioned medium or secretome: In addition to extracellular vesicles, these materials contain cytokines, growth factors, metabolites, and soluble proteins. Their effects cannot be attributed entirely to exosomes.

④ Plant-derived extracellular vesicles or vesicle-like nanoparticles: Their origins, membrane compositions, and formation mechanisms differ from those of mammalian-cell exosomes. They should be defined according to their actual material composition.

⑤ Engineered vesicles, extracellular vesicle mimetics, or liposomes: These may be prepared through cell extrusion, artificial lipid assembly, or fusion between natural vesicles and liposomes. They should not be directly described as natural exosomes.

2 How the Source of Exosomes Affects Ingredient Properties

2.1 Major Biological Sources

Extracellular vesicles derived from mesenchymal stromal cells are widely used in tissue-repair research. However, a mesenchymal stromal cell origin alone does not demonstrate that an ingredient has anti-inflammatory, collagen-promoting, or anti-aging effects. Vesicles produced from different tissue sources, donors, and culture methods may differ substantially in composition and potency.

2.2 Source Cells and Culture Conditions Affect Extracellular Vesicle Cargo

Cells can selectively load certain proteins, lipids, and RNAs into vesicles. Vesicle cargo is influenced by the following factors:

① Cell type and tissue of origin;

② Donor age, health status, and individual variability;

③ Cell passage number and degree of cellular senescence;

④ Culture medium and serum-containing or serum-free culture conditions;

⑤ Oxygen concentration, nutrient availability, and cell density;

⑥ Ultraviolet exposure, inflammation, hypoxia, and oxidative stress;

⑦ Two-dimensional culture, three-dimensional culture, or bioreactor-based culture.

The functional state of the source cells may alter the direction of vesicle activity. For example, vesicles produced by healthy cells may carry signals that support tissue homeostasis, whereas vesicles produced by senescent, inflamed, or abnormal cells may carry pro-inflammatory, profibrotic, or other unintended signals.

3 Preparation, Purification, and Quality Control of Exosomes

3.1 Preparation Process from Cell Culture Medium to the Final Ingredient

A relatively complete preparation process can be summarized as follows:

Source-cell selection and establishment of a cell bank → controlled culture → collection of conditioned medium → removal of cells and large debris → vesicle concentration → vesicle purification → buffer exchange and formulation → release testing and stability evaluation

Concentration and purification serve different purposes:

① Concentration increases the number of particles per unit volume but may also concentrate impurities.

② Purification reduces the levels of free proteins, lipoproteins, nucleic acids, culture-medium components, and other non-vesicular particles.

Polymer precipitation, ultrafiltration, and tangential flow filtration may be used for concentration, but none of these methods alone demonstrates that highly purified exosomes have been obtained.

3.2 Comparison of Major Isolation and Purification Techniques

A single technique usually cannot simultaneously provide high recovery, high purity, and good scalability. A commonly used combined process is:

TFF concentration and diafiltration → SEC or other chromatographic polishing → sterile filtration or aseptic filling, depending on the intended use and process-validation results → formulation

A combined process does not automatically ensure ingredient quality. Membrane-retention range, chromatography media, flow rate, temperature, buffer composition, and sample loading may all affect vesicle recovery, subpopulation composition, and biological activity.

3.3 Quality Attributes That Should Be Confirmed for Exosome Ingredients

A reasonable assessment of exosome quality should cover four levels:

The molecular characteristics of extracellular vesicles should be evaluated using a combination of different marker classes. Membrane-associated EV proteins, EV-related cytosolic proteins, and potentially co-isolated non-vesicular components should generally be assessed together to determine whether the sample contains EVs and to evaluate its purity. Specific markers should be selected according to the cell source and isolation method. Detection of CD9, CD63, or CD81 alone does not prove that a sample consists of exosomes or that it is highly pure.

Nanoparticle tracking analysis can measure particle size and concentration within a certain range but cannot reliably distinguish exosomes from lipoproteins, virus-like particles, or protein aggregates. A high particle count may indicate high vesicle recovery, but it may also reflect a high level of non-vesicular particles.

3.4 Formulation Stability Determines Whether Activity Is Retained at the Time of Use

Exosome membranes and their cargo may be affected by temperature, pH, ionic strength, oxidation, shear stress, freeze–thaw cycles, and formulation components. Common changes include:

① Membrane rupture or increased permeability;

② Particle aggregation and increased particle size;

③ Changes in the conformation of surface proteins;

④ Degradation of RNA and proteins;

⑤ Reduced vesicle uptake or functional activity.

Lyophilization can reduce dependence on continuous low-temperature cold-chain storage, but the lyophilization process itself may cause membrane damage and aggregation. Protectants such as sucrose and trehalose may improve post-reconstitution integrity in some systems, but their protective effects depend on the vesicle source, buffer composition, lyophilization cycle, and residual moisture.

Stability in the bulk ingredient solution does not demonstrate that the vesicles will remain stable after incorporation into the final skincare formulation. Surfactants, high concentrations of ethanol, preservatives, fragrances, extreme pH values, and repeated opening of the package may affect vesicle structure. Efficacy and stability should therefore be evaluated in the final formulation, final packaging, and over the labeled storage period.

4 Basic Mechanisms by Which Exosomes May Act on the Skin

4.1 Biological Effects Require a Continuous Delivery Process

The activity of exosomes can be summarized as follows:

Vesicles remain intact and active in the formulation → reach the target region of the skin → contact target cells → surface molecules trigger signaling or the vesicles are internalized → active cargo reaches its site of action → cellular signaling, gene expression, or metabolism is altered → tissue-level changes occur

In cell-culture experiments, vesicles are generally added directly to the culture medium and can readily contact recipient cells. Topical skincare products must additionally undergo storage, application, residence on the skin surface, and passage through the stratum corneum barrier. Consequently, the actual exposure dose differs substantially between cell-culture experiments and topical use.

4.2 Modes of Interaction Between Exosomes and Skin Cells

Exosomes may affect keratinocytes, fibroblasts, immune cells, and vascular endothelial cells through the following mechanisms:

① Binding of surface ligands to receptors

Integrins, tetraspanins, glycoproteins, and lipids on the vesicle surface may bind to cell-membrane receptors or proteoglycans and directly initiate cellular signaling. Such effects do not necessarily require internal cargo to enter the cell.

② Cellular endocytosis

Vesicles may enter cells through clathrin-mediated endocytosis, caveolae-associated endocytosis, macropinocytosis, or phagocytosis. The specific route depends on the vesicle source, surface composition, and recipient-cell type.

③ Intracellular trafficking

Internalized vesicles generally enter the endosomal system first. Some are transported to lysosomes and degraded, whereas others may undergo membrane fusion or escape from the endosome.

④ Release of functional cargo

Some proteins or RNAs may enter the cytoplasm and participate in biological processes. However, “cellular uptake of vesicles” is not equivalent to “effective release of vesicle cargo.” Quantitative studies have shown that functional cargo-delivery efficiency may be substantially lower than particle-uptake efficiency. Endosomal retention and lysosomal degradation are important limiting factors.

The theoretical process through which miRNA cargo produces post-transcriptional regulation can be summarized as follows:

Exosome uptake → endosomal processing and partial cargo release → entry of miRNA into the cytoplasm → loading into Argonaute/RNA-induced silencing complexes → pairing with target mRNA → mRNA degradation or translational repression → reduced expression of the target protein

This process can produce a measurable effect only if the vesicles contain a sufficient quantity of functional miRNA and the miRNA successfully reaches the cytoplasm of the recipient cells.

4.3 Regulation of Inflammatory Signaling

Ultraviolet radiation, barrier damage, and oxidative stress may activate nuclear factor kappa B (NF-κB) signaling:

Ultraviolet radiation, reactive oxygen species, or inflammatory receptor activation → activation of IκB kinase → phosphorylation and degradation of IκB → translocation of NF-κB into the nucleus → increased expression of inflammation-related genes such as interleukin-6 and tumor necrosis factor-α

In some studies of extracellular vesicles derived from mesenchymal stromal cells, vesicular proteins, lipids, or regulatory RNAs have been associated with changes in NF-κB signaling, mitogen-activated protein kinase (MAPK) signaling, and macrophage function, potentially reducing excessive inflammatory responses.

This effect is not shared by all exosomes. When source cells are exposed to inflammatory stimuli, their vesicles may instead enhance inflammatory signaling. Whether a particular exosome ingredient has anti-inflammatory activity must be established through cytokine assays, receptor-signaling studies, and dose–response experiments using the specific ingredient.

4.4 Effects on Oxidative Stress

Ultraviolet irradiation can increase reactive oxygen species (ROS) in skin cells, including superoxide anion radicals (O₂•⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH). Some of the basic reactions involved are:

O₂ + e⁻ → O₂•⁻

2 O₂•⁻ + 2 H⁺ —SOD→ H₂O₂ + O₂

2 H₂O₂ —CAT→ 2 H₂O + O₂

Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + •OH

SOD denotes superoxide dismutase, and CAT denotes catalase. When ROS generation exceeds the cellular capacity for removal, it can cause lipid peroxidation, DNA and protein damage, and increased inflammation and collagen degradation.

Extracellular vesicles from certain sources may regulate nuclear factor erythroid 2-related factor 2 (Nrf2) signaling in experimental models:

Oxidative stress alters the state of Keap1 → Nrf2 degradation decreases and Nrf2 enters the nucleus → Nrf2 binds to antioxidant response elements (AREs) → increased expression of antioxidant-related genes such as HO-1, NQO1, and GCLC

The effects of extracellular vesicles on oxidative stress generally involve regulation of cellular antioxidant-defense systems rather than direct scavenging of free radicals. These effects depend on the vesicle source, cargo composition, dose, and experimental conditions and must be verified for each specific ingredient.

4.5 Effects on Fibroblasts and Collagen Metabolism

Ultraviolet radiation can increase the expression of matrix metalloproteinases (MMPs) through signaling pathways such as activator protein 1 (AP-1) and NF-κB. MMP-1 can initiate cleavage of type I and type III fibrillar collagen, after which other MMPs further degrade the collagen fragments, disrupting the dermal collagen network.

The process can be summarized as follows:

Increased ultraviolet exposure or ROS → activation of AP-1/NF-κB → increased expression of MMP-1 and related enzymes → accelerated degradation of type I and type III collagen → damage to the dermal extracellular matrix

Some extracellular vesicles may reduce MMP expression in fibroblast or animal models or affect transforming growth factor beta (TGF-β)/Smad signaling:

TGF-β binds to its receptor → phosphorylation of Smad2/3 → formation of a Smad2/3–Smad4 complex → translocation into the nucleus → regulation of COL1A1, COL1A2, COL3A1, and related genes → changes in collagen synthesis

Vesicles may also affect fibroblast survival, proliferation, and migration through phosphoinositide 3-kinase (PI3K)/Akt and MAPK/ERK signaling.

An increase in collagen-related gene or protein expression does not necessarily mean that well-organized dermal collagen with normal mechanical properties has formed. Evaluation of anti-wrinkle effects also requires assessment of collagen maturation, fiber architecture, tissue mechanics, and changes in human skin.

4.6 Effects on Epidermal Repair and Angiogenesis

Certain extracellular vesicles may affect the following processes in wound models:

① Keratinocyte migration and re-epithelialization;

② Fibroblast migration and extracellular matrix deposition;

③ Macrophage activity and the local inflammatory state;

④ Vascular endothelial growth factor-related signaling and neovascularization;

⑤ Remodeling of the wound extracellular matrix.

The simplified process is:

Vesicles interact with cells in the damaged area → regulation of inflammation, cell migration, and angiogenic signaling → changes in re-epithelialization and granulation-tissue formation → progression to the matrix-remodeling stage

Wound models lack an intact stratum corneum barrier, allowing vesicles to contact viable cells directly. Results from wound-healing studies therefore cannot be used directly as evidence of anti-aging efficacy after topical application to intact skin.

5 Can Exosomes Penetrate the Skin Barrier?

5.1 The Intact Stratum Corneum Restricts Passive Vesicle Penetration

The stratum corneum consists of corneocytes and intercellular lipids and forms a strong barrier to hydrophilic macromolecules and nanoparticles. Exosomes are generally membrane-enclosed particles ranging from tens to hundreds of nanometers in size, making them substantially larger than small molecules suitable for passive transdermal diffusion.

After conventional topical application, exosomes may be distributed mainly in:

① The skin surface;

② The superficial layers of the stratum corneum;

③ Skin furrows and the surfaces of intercellular spaces;

④ Follicular openings and the upper portions of hair follicles.

Hair follicles can serve as local reservoirs for nanoparticles. However, follicular deposition does not mean that intact vesicles have entered the viable epidermis or dermis. Current evidence is insufficient to demonstrate that intact exosomes in conventional skincare formulations can reproducibly cross the intact human stratum corneum at a dose sufficient to produce a biological effect.

5.2 Transdermal Studies Are Susceptible to Detection-Method Artifacts

Many studies label vesicles with lipophilic fluorescent dyes. These dyes may dissociate from the vesicle membrane, form dye aggregates, or transfer to skin lipids. Observation of a fluorescent signal alone does not demonstrate that intact vesicles have reached the corresponding depth of the skin.

More reliable transdermal studies should confirm all of the following:

① The label remains associated with intact vesicles;

② Vesicle structures remain present at different depths of the skin;

③ The detected signal is not derived from free dye or vesicle fragments;

④ The quantity reaching the target layer is sufficient to produce a biological effect.

5.3 Microneedling and Laser Treatment Change the Conditions of Product Use

Microneedling, radiofrequency microneedling, and fractional laser treatment can create microchannels in the stratum corneum, enabling exosomes and other formulation components to contact the viable epidermis or dermis more readily:

Formation of microchannels → weakening of the stratum corneum barrier → increased contact between exosomes and viable skin cells

When the barrier is weakened, non-target components in the product may also enter deeper skin layers. Microbial contamination, bacterial endotoxins, residual culture-medium proteins, and process-related impurities remaining after purification may all increase the risk of infection, irritation, or abnormal inflammatory responses.

Conventional topical cosmetics are generally subjected to quality control and safety assessment based on application to intact skin. Their requirements for microbial control, endotoxin control, and sterility differ from those for preparations used after microneedling, laser treatment, or other invasive procedures. A product labeled for topical use therefore cannot be assumed to be suitable for application after microneedling or laser treatment and should not be used on visibly damaged or open skin.

Microneedling and laser treatment themselves initiate inflammation, cell migration, and collagen remodeling. Skin improvements observed after combined treatment may result from the procedure itself, the exosome formulation, or both and cannot be attributed entirely to exosomes.

6 Safety Concerns, Regulatory Considerations, and Precautions for Use

6.1 Safety Risks Arise Primarily from the Ingredient and Manufacturing Process

Exosomes do not contain intact living cells, but they remain complex, biologically active particles. Major risks include:

① Incorrect source-cell identity, cellular senescence, or genetic abnormalities;

② Risk of transmission of donor-associated pathogens;

③ Bacterial, fungal, mycoplasmal, or viral contamination;

④ Endotoxin and culture-medium residues;

⑤ Residual serum proteins, antibiotics, and growth factors;

⑥ Residual host-cell proteins and DNA;

⑦ Residual polymer precipitants or other processing aids;

⑧ Batch-to-batch variation in composition and potency;

⑨ Unintended inflammatory, immune, or coagulation-related effects.

6.2 The Route of Administration Changes the Level of Risk

Different routes of use result in different levels of tissue exposure:

Topical application to intact skin → application to barrier-impaired skin → use after microneedling or laser treatment → intradermal or subcutaneous injection → intravenous or other systemic administration

As the degree of invasiveness increases, requirements for sterility, endotoxin control, viral safety, dosing, biological potency, and impurity control also increase.

The U.S. Food and Drug Administration (FDA) has received reports of serious adverse events involving patients who used unapproved therapeutic products claimed to contain exosomes. The FDA has stated that exosome products intended for medical purposes, such as the treatment of disease, are generally regulated as drugs or biological products and must undergo the corresponding premarket review and approval process. At present, the FDA has not approved any exosome product for the treatment of disease.

6.3 Requirements Applicable to Cosmetics in China

The service guidelines and frequently asked questions for the filing of new cosmetic ingredients, published through the government service portal of China’s National Medical Products Administration, state that substances prohibited under the Safety and Technical Standards for Cosmetics do not qualify as new cosmetic ingredients. These prohibited substances include human cells, tissues, and products of human origin. The guidelines also state that ingredients claimed to “activate cells,” “regenerate cells,” “promote healing,” or exert similar medical effects fall outside the statutory definition of cosmetics.

Based on these publicly available rules, human-derived extracellular vesicles clearly have a human biological origin and face substantial regulatory barriers when considered for use as ingredients in conventional cosmetics. Plant-derived or other non-human vesicles must also be evaluated according to their actual source, preparation method, composition, safety profile, and regulatory status. A non-human origin alone does not establish that a material is compliant for cosmetic use.

7 Representative Reagents and Materials for Research on Exosome Preparation, Quality Assessment, and Mechanisms of Action

Table 1. Products Related to Exosome Isolation and Purification, Buffering, and Formulation Stability

This group of products covers precipitation-based enrichment, density-gradient purification, buffer preparation, freeze–thaw protection, lyophilization bulking, and formulation-stability studies involving extracellular vesicles. Conditions should be selected according to the exosome source, isolation process, and subsequent analytical methods.

Table 2. Products Related to Exosome Fluorescence Tracking and Oxidative-Stress Detection

This group of products is used for vesicle-membrane labeling, cell and vesicle tracking, observation of cellular uptake, and validation of mechanisms involving reactive oxygen species. Fluorescence-tracking experiments should include controls for free dye, dye aggregates, and unlabeled samples to avoid misinterpreting dye transfer as delivery of intact vesicles.

Table 3. Products Related to Exosome Biogenesis, Release, Uptake, and Intracellular Trafficking

This group of products is mainly used to interfere with ceramide metabolism, the cytoskeleton, cholesterol-dependent membrane structures, clathrin-mediated endocytosis, macropinocytosis, dynamin activity, and endosome–lysosome trafficking. Chemical inhibitors may affect multiple cellular processes simultaneously. Experimental findings should therefore be validated using cell-viability assessments, genetic interventions, and multiple uptake-detection methods.

Table 4. Products Related to Mechanistic Studies of Skin Inflammation, Repair, and Extracellular-Matrix Regulation

This group of products is used to determine whether exosomes influence inflammatory responses, cell migration, collagen metabolism, and skin repair through signaling pathways involving transforming growth factor beta, phosphoinositide 3-kinase, nuclear factor kappa B, matrix metalloproteinases, and related mechanisms.

Note: The products listed above are representative Aladdin products for scientific research and formulation studies. Additional product specifications, grades, and certificates of analysis may be retrieved from the Aladdin website using the product name, CAS number, or catalog number.

References

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