Plant-derived exosome-like nanovesicles: a novel therapeutic perspective for skin diseases

Abstract

Plant-derived exosome-like nanovesicles (PENs) are emerging as a unique class of bioactive nanocarriers with transformative potential in dermatology. Characterized by intrinsic biocompatibility, the ability to traverse biological barriers, and a molecular cargo rich in lipids, proteins, nucleic acids, and phytochemicals, PENs exert multi-target effects, including anti-inflammatory and antioxidant activities, modulation of melanogenesis, and promotion of tissue repair. In this review, we synthesize current advances in PEN biology, encompassing their biogenesis, molecular architecture, and cutting-edge methods for isolation, purification, and characterization. We also highlight translational progress in treating skin aging, alopecia, pigmentation disorders, and impaired wound healing, while critically addressing challenges that impede clinical adoption, such as toxicology, stability, delivery efficiency, manufacturing scalability, and regulatory compliance. By integrating mechanistic insights with translational strategies, PENs are poised to redefine therapeutic and cosmetic paradigms in skin health, offering a rare convergence of natural origin, molecular precision, and clinical promise.

Graphical abstract

Introduction

The skin, acknowledged as the largest organ in the human body, accounts for approximately 16% of total body weight [1]. This system is characterized by a multilayered barrier comprising three distinct layers: the epidermis, dermis, and hypodermis. This intricate hierarchical structure plays an essential role in maintaining homeostasis; however, it presents significant challenges for drug delivery. The epidermis, which forms the outermost layer of the skin, serves as a primary protective barrier against pathogenic microorganisms, ultraviolet (UV) radiation, and a broad spectrum of environmental stressors [2]. Despite this protective role, it substantially restricts the transdermal permeation of most pharmaceutical agents, thereby posing a major obstacle to their effective delivery. Even when the structural integrity of the skin barrier is compromised, facilitating drug passage through the stratum corneum remains a formidable challenge. Beneath the epidermis lies the dermis, which ranges from 1 to 4 mm and is primarily composed of collagen fibers and fibroblasts that confer structural support, elasticity, flexibility, and resilience [3]. The papillary dermis is distinguished by its abundant vascular network, nerve endings, sebaceous glands, and sweat glands. In contrast, the deeper reticular dermis encompasses blood vessels, lymphatic channels, hair follicles, and sweat ducts, all of which play critical roles in transdermal drug delivery (TDD). Although the dermis contains a dense capillary network, effective systemic drug absorption necessitates prior traversal of the epidermal barrier. The hypodermis, also known as subcutaneous adipose tissue, constitutes the innermost layer of the skin and is primarily composed of adipocytes situated within a matrix of glycoproteins and glycosaminoglycans [4]. This layer fulfills several essential functions, including thermal insulation, mechanical cushioning, energy storage, and the attachment of the skin to the underlying musculature or skeletal structures[5].

The increasing incidence of skin diseases poses a major public health challenge. Factors such as genetic predisposition, lifestyle, nutritional status, UV exposure, photosensitivity, and pollution impair skin function, weaken barrier integrity, and deplete essential components, leading to aging, alopecia, pigmentary disorders, and poor wound healing. Current treatments include topical products, laser therapy, drugs, surgery, and cell-based approaches [6]. However, these methods are limited by poor drug penetration, adverse effects, and low patient adherence, making comprehensive repair difficult. A deeper understanding of disease mechanisms and more effective strategies are critical. Recent advances highlight exosome-based therapies as promising approaches for restoring skin function and treating diverse dermatological conditions.

In recent years, plant-derived exosome-like nanovesicles (PENs) have garnered significant attention as promising nanodrug delivery systems. This interest is attributed to their wide availability, natural composition, ease of large-scale extraction from green resources [7, 8], small size, strong tissue permeability, negative zeta potential, prolonged circulation time, similarity to cell membranes, and high biocompatibility and stability. Additionally, PENs exhibit physicochemical stability across various pH values and temperatures [9, 10]. PENs are a type of extracellular vesicle with a diameter ranging from 50 to 500 nm enriched with a variety of bioactive components, including lipids, proteins, RNA, and other effective small molecules [11–13]. They are derived from a wide range of sources, including fruits [14–18], vegetables [19, 20], seeds [21, 22], rhizomes [23], leaves [24, 25], flowers [26, 27], tea [28, 29], and nuts [30]. Exosomes have been isolated from various plant cells. Research on PENs continues to reveal their significant biological functions, including antioxidation, anti-inflammatory properties, anti-aging effects, prevention of hair loss, skin whitening, and promotion of wound healing [31]. PENs also demonstrate significant potential as delivery vehicles in therapeutic applications. The flexible lipid bilayer architecture allows for interactions with the lipids of the stratum corneum, thereby promoting the distribution of drugs within the skin. This interaction, combined with their intrinsic biological characteristics, synergistically improves therapeutic effectiveness.

This review systematically synthesizes the biological foundations, mechanisms of action, and functional roles of PENs within the context of dermatological therapy. Emphasizing four principal domains—anti-aging, anti-alopecia, skin whitening, and wound healing—it integrates recent findings from in vitro experiments, animal studies, and preclinical investigations. The analysis evaluated the therapeutic potential and clinical benefits of PENs in enhancing skin health and managing various dermatoses, while also outlining current limitations. A comprehensive understanding of the critical functions of PENs is expected to provide novel insights and inform therapeutic strategies in dermatology, thereby advancing their clinical application as innovative treatment modalities.

Basic characteristics of pens

Distinctions between pens and animal-derived exosome-like nanovesicles

As crucial nanocarriers for intercellular communication and biological delivery, animal exosomes and PENs have garnered significant attention in recent years. Both entities contain proteins, lipids, and RNA, demonstrating structural similarities and shared biological functionalities [32, 33]. However, there are notable differences in their origin, composition, in vivo behavior, and potential applications (Fig. 1). Exosomes derived from animal sources are characterized by smaller particle sizes, ranging from 30 to 150 nm, and are enriched with proteins associated with membrane fusion, as well as a variety of nucleic acid molecules, including mRNAs, miRNAs, and long noncoding RNAs (lncRNAs). These features enhance the effective delivery of hydrophobic pharmaceuticals. However, the use of these xenogeneic exosomes poses risks related to immunogenicity and the potential for viral transmission [10, 34]. In contrast, PENs exhibit a broader range of biological origins and possess larger particle sizes, typically ranging from 50 to 500 nm. They encompass bioactive components specific to plants, as well as microRNAs. The presence of structural phytosterols enhances their immunomodulatory and anti-inflammatory effects. Furthermore, their stable membrane lipid composition contributes to improved gastrointestinal stability and targeting specificity, making them particularly suitable for oral administration [35], although their distribution beyond the gastrointestinal tract remains limited. Engineered PENs achieve precise delivery through surface modification with targeting ligands. Following systemic administration, both types of nanocarriers are distributed primarily to hepatic, renal, splenic, and gastrointestinal tissues following systemic administration [36]. Notably, PENs exhibit enhanced biocompatibility and engineering capabilities, indicating significant potential for diverse applications in anti-infective, anti-fibrotic, and anti-tumor therapeutic interventions [37]. These unique attributes confer substantial advantages to PENs over exosomes derived from animal sources, particularly in the context of clinical application. Furthermore, these findings provide a theoretical foundation for investigating PENs in the treatment of dermatological conditions.

Fig. 1.

Differences between PENs and animal-derived exosomes. PENs exhibit a larger particle size, contain plant-specific bioactive components and microRNAs, and possess a stable membrane structure (Figure created using BioRender)

Biogenesis of pens

In the 1960 s, researchers first observed through electron microscopy that carrot cells can secrete vesicles [38]. In 2009, the isolation of exosome-like vesicles from plants, specifically sunflower seeds, was reported for the first time [21].

Compared with animal-derived exosome-like nanovesicles, research on PENs remains relatively limited, particularly regarding their biogenesis mechanisms. Previous studies have proposed that these organelles may form via three pathways: the multivesicular body (MVB) pathway, the EXPO (exocyst-positive organelle) pathway, and the vacuolar pathway [39].

MVB pathway: This pathway has been well studied in plant cells and recognized as the primary PEN generation mechanism. It centers on late endosomes (MVBs) formed by endocytosis and initiated through plasma membrane invagination [40]. This process results in the formation of early endosomes, which mature via interaction with the trans-Golgi network (TGN) into late endosomes. MVBs contain intraluminal vesicles (ILVs) that are 30–100 nm in diameter and selectively package cytoplasmic components—lipids, proteins, nucleic acids, and small molecules—into ILVs [41]. ILV formation is mediated by the ESCRT mechanism, which drives MVB membrane curvature. Upon plasma membrane fusion, MVBs release exosomes, which are functional, uniformly sized vesicles. These exosomes enter target cells via receptor binding or membrane fusion to deliver cargo.

EXPO pathway: This is a plant-specific secretory mechanism. EXPOs are double-membraned spherical structures resembling autophagosomes, identified in Arabidopsis thaliana [42]. Although structurally similar to autophagosomes and capable of fusing with the plasma membrane to release single-membrane vesicles into the cell wall, these vesicles represent EXPO-secreted exosomes. The pathway comprises intracellular EXPO formation, plasma membrane fusion, and subsequent PEN release [43]. Marker proteins such as Exo70H1 and Exo70B2 are key to EXPO-mediated trafficking, are associated with plant innate immunity, and induce pathogen-triggered immunity (PTI), enhancing Arabidopsis resistance to pathogens [44]. Thus, EXPO-derived vesicles facilitate intercellular communication and plant defense signaling [42].

Vacuolar pathway: This pathway is linked to disease resistance and defense responses, where vacuoles secrete hydrolases and antimicrobial proteins extracellularly to combat pathogens [45]. Plant vacuoles are multifunctional organelles responsible for storage, degradation, and homeostasis maintenance. Small vacuoles (SVs) form through the fusion of MVBs, whereas central vacuoles develop via the maturation and fusion of SVs. Studies indicate that vacuole formation involves the transformation of MVBs into SVs, which subsequently fuse to form central vacuoles [43]. Thus, the vacuolar trafficking pathway may be involved in PEN biogenesis; however, the underlying mechanisms require further investigation [46].

Structure and composition of pens

Exosomes transport a diverse array of active components, typically composed of lipids, proteins, nucleic acids, and specific small molecules. The first three components serve as critical criteria for quality control [47]. The content, size, and membrane composition of exosomes are highly heterogeneous and are dynamically influenced by the cellular source, cellular state, and environmental conditions. The biogenesis and structural characteristics of PENs are illustrated in Fig. 2.

Fig. 2.

The biogenesis and composition of exosomes occur through three distinct pathways: (1) the vacuolar pathway: in this pathway, vacuoles containing hydrolytic enzymes and defensive components merge with the plasma membrane, thereby releasing these enzymes and proteins to combat pathogen invasion during infection; (2) the multivesicular bodies (MVBs) pathway: MVBs are derived from the trans-Golgi network (TGN) or early endosomes. Within MVBs, intraluminal vesicles (ILVs) selectively encapsulate various cargo molecules, including DNA, RNA, and lipids. MVBs subsequently fuse with the plasma membrane, resulting in the release of exosomes; (3) the exocyst-positive organelle (EXPO) pathway: this pathway involves double-membrane EXPO structures, which are similar to those of autophagosomes. These structures directly fuse with the plasma membrane to release vesicles into the extracellular space. PENs consist of lipids, proteins, nucleic acids, and specific small molecules (Figure created with BioRender)

Lipids

Lipids play crucial roles in the bilayer structure of PENs, influencing their functionality and capacity for cellular uptake. PENs are rich in various lipids, including phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), diacylglycerol (DAG), triacylglycerol (TAG), digalactosyl diacylglycerol (DGDG), and monosemi-lactosyldiacylglycerol (MGDG) [48]. PA is present at the highest concentration in PENs and primarily facilitates cell proliferation, signal transduction, and the uptake of PENs, whereas PE plays a significant role in regulating membrane curvature. The types and concentrations of lipids in plant exosomes vary across different fruits. Notably, all the examined PENs lack cholesterol to date. We hypothesize that the lipid composition of PENs influences their absorption and distribution within the skin. Studies have demonstrated that exosomes derived from ginger, referred to as ginger-derived exosome-like nanovesicles (GELNs), are preferentially taken up by members of the Lactobacillus family within the gut microbiota. This specific uptake mechanism is closely associated with the structural characteristics of the lipid components present in the exosomes [49].

Protein

PENs are typically characterized by their low protein concentrations, with their proteomic profiles primarily consisting of cytosolic and transmembrane proteins [39]. Compared with the protein composition of animal exosomes, the proteins in PENs exhibit a reduced variety and are present in lower quantities. A proteomic map was generated to elucidate the uptake mechanism of these proteins [50]. Studies have demonstrated that surface proteins derived from garlic exosomes facilitate their internalization by binding to the CD98 glycoprotein on the surface of human liver cancer cells (HepG2). Blocking the CD98 receptor on HepG2 cells resulted in a 47.01% reduction in the uptake of garlic exosomes, confirming the critical role of their surface lectin-like proteins in cellular uptake [51]. Proteomic analysis of exosomes derived from bitter melon has revealed significant antioxidant properties, suggesting their potential therapeutic efficacy in alleviating skin conditions associated with oxidative stress [52]. Currently, among transmembrane proteins, CD63, CD81, and CD9 have been identified as protein markers of mammalian cells. Although the protein composition and functions of PENs have been partially characterized, specific markers remain inadequately defined, complicating the identification of their source, purity assessment, and evaluation of their biomedical functions.

Nucleic acid

The nucleic acids enclosed in exosomes play essential roles in intercellular communication and signal transduction, with microRNAs (miRNAs) serving as key mediators in exosome-based therapies [53]. These miRNAs can regulate protein expression posttranscriptionally in both plants and animals, and they can act across species to modulate human mRNA expression, participating in inflammation, immune regulation, and tumor-related pathways [54]. Furthermore, PENs can be taken up by gut microbiota, where their RNA cargo modulates microbial composition and host immunity, contributing to skin homeostasis through the gut–skin axis. PENs can be absorbed by gut microbiota, influencing its composition and the functions of the host through the RNA they carry. GELN-enriched miRNAs demonstrate anti-inflammatory and physiological regulatory effects by targeting human digestive organs through cross-species mechanisms. These miRNAs reduce the expression of pro-inflammatory cytokines such as NF-κB, IL-6, IL-8, and TNFα, thereby mitigating lipopolysaccharide (LPS)-induced inflammation and contributing to the regulation of inflammatory and cancer-related pathways [23]. These mechanisms are highly relevant for the regulation of the skin barrier. Recent investigations have revealed for the first time that osa-miR164d within GELNs can markedly reduce colonic inflammation by targeting Table 1, which inhibits the NF-κB signaling pathway and alters macrophage polarization. Additionally, researchers have developed biomimetic exosomes for the targeted delivery of this miRNA, achieving efficient immune regulation and providing a novel strategy for the delivery of PEN-mediated miRNAs. This approach suggests potential therapeutic avenues for treating inflammatory diseases, including those affecting the skin [55]. PENs can effectively mitigate inflammation and cellular damage by inhibiting the expression of key viral proteins (Nsp12 and S protein), providing valuable insights for the treatment of COVID-19, and may offer novel therapeutic strategies for viral-associated cutaneous disorders such as COVID-19-related dermatological injuries [56].

Table 1.

Main separation methods for pens: advantages and disadvantages

Isolation methods	Advantages	Disadvantages	References
Ultracentrifugation	The classical method is often used as the basis for separation, and the experimental conditions are mature	The time is long, the equipment requirements are high, the particles are easy to aggregate or destroy, and the purity is relatively low	[62, 63]
Sucrose density gradient Centrifugal separation	The separation is accurate and does not affect the activity	The preparation process is complicated, the yield is low, and it is easy to be affected by centrifugal parameters	[64–66]
Polymer precipitation	Easy to operate, fast, suitable for mass production	Impurities such as proteins are easy to coprecipitate, which may affect the biological characteristics of exosomes	[67, 68]
Exclusion chromatography	High purity, maintaining its structural and functional integrity	Provide low output, high equipment requirements, complex operation, not easy to scale production	[69, 70]
Microfluidic technology	Miniaturization, automation and integration, compatible with a variety of separation methods, efficient capture and resolution of exosomes, providing a stable high-throughput technology platform	The equipment is expensive, the experimental conditions and the application range are limited	[71]

Small active molecule products

PENs contain a variety of homologous small-molecule active components derived from plants that play crucial roles in various biological functions. These components are typically analyzed using liquid chromatography-mass spectrometry. The primary active components found in lemon exosomes include hesperidin, naringin, neohesperidin, flavonoids, limonin, and citric acid, which exhibit antioxidant, depigmenting, and anti-inflammatory properties. Notably, hesperidin has been demonstrated to reduce melanin synthesis by suppressing tyrosinase expression [57], suggesting its therapeutic potential for hyperpigmentary dermatoses. Exosomes derived from yam, although lacking conventional saponin components, have been shown to activate the BMP-2/p38/Runx2 signaling pathway, thereby promoting osteoblast differentiation [58]. This pathway also plays a crucial role in skin wound healing and the regulation of hair follicle stem cells. Studies have demonstrated that BMP-2 can induce autophagy and differentiation of hair follicle stem cells by upregulating PTEN expression, thus enhancing epidermal reconstruction and hair regeneration. Moreover, the periodic activation of this pathway during the early stages of injury has been identified as a key mechanism in the regulation of skin regeneration and the activation of hair follicles [59]. Recent studies have indicated that bioactive small molecules are not actively incorporated into PENs but rather accumulate within vesicle membranes through passive hydrophobic interactions. Molecules with greater lipophilicity show increased affinity for membrane binding and enhanced stability, while hydrophilic compounds exhibit a reduced capacity for retention [32].As natural nanocarriers, PENs improve the aqueous solubility and transmembrane permeability of these bioactive molecules, thereby providing a solid basis for their therapeutic use in dermatological conditions.

Separation of pens and identification

The study of PENs is fundamentally grounded in their effective isolation and accurate characterization. Given the complex nature of plant-derived matrices, exosomes obtained from various botanical tissues exhibit significant heterogeneity in terms of their origin, structural composition, and functional characteristics. Consequently, extraction protocols must be meticulously designed to preserve both purity and biological integrity. The adoption of advanced separation and purification technologies not only guarantees methodological precision for subsequent functional analyses but also establishes a robust material foundation for their potential applications in dermatological therapies. Currently, the predominant techniques for the isolation and purification of PENs include ultracentrifugation methods [60],density gradient centrifugation [61], polymer precipitation, SEC, and microfluidic technology. We summarized and compared different extraction methods for PENs (see Fig. 3; Table 1), aiming to provide a reference for the standardization of extraction protocols and subsequent applications.

Fig. 3.

Methods for the extraction, isolation, and purification of PENs. (A) Ultracentrifugation. (B) Sucrose density gradient centrifugal separation. (C) Polymer precipitation. (D) Exclusion chromatography. (E) Microfluidic technology. (F) High-pressure processing combined with ultracentrifugation and aqueous two-phase extraction. (G) Electrophoresis–dialysis (ELD). (H) Enzymatic digestion (cellulase + pectinase). Figure created with BioRender

Separation methods

Ultracentrifugation

Ultracentrifugation is widely utilized and regarded as the “gold standard” for exosome preparation because of its straightforward operational procedure, low contamination risk, and lack of reagent use, which collectively facilitate the production of high yields of exosome-like nanoparticles [62]. This method leverages the differences in sedimentation rates among sample components subjected to varying centrifugal forces to isolate exosomes. Initially, fresh plant tissue was homogenized using a high-speed grinder, followed by filtration to eliminate impurities. Coarse debris was removed through low-speed centrifugation. After the supernatant was collected, it underwent multiple rounds of centrifugation and was ultimately subjected to high-speed centrifugation at 100,000–120,000×g for 90 min. The precipitated nanoparticles were then collected and resuspended in phosphate-buffered saline (PBS). Exosomes extracted from Polygonatum sibiricum were obtained through ultracentrifugation, and their mechanisms of action against breast cancer were investigated via a comprehensive approach that integrated proteomics, metabolomics, and network pharmacology [72]. Exosomes extracted from Rabdosia rubescens via ultracentrifugation were incorporated into composite hydrogels to evaluate their therapeutic efficacy in promoting the healing of oral mucositis induced by chemoradiotherapy [73]. Exosomes derived from Momordica charantia L. through ultracentrifugation demonstrated cardioprotective properties against doxorubicin-induced cardiotoxicity [74]. Exosomes obtained from Koshihikari rice bran suspensions via a series of sequential centrifugations, 0.45 μm filtration, and ultracentrifugation significantly inhibited the proliferation of colon adenocarcinoma cells and induced apoptosis [75]. Repeated excessive centrifugation not only compromises the integrity of PENs and diminishes their biological efficacy [63], but also constrains their broader application owing to the stringent requirements for laboratory conditions, the necessity for specialized techniques, and the associated high costs.

Sucrose density gradient centrifugation

Extended high-speed operations in ultracentrifugation can result in the aggregation and precipitation of proteins and nucleic acids. Consequently, sucrose density gradient centrifugation is frequently employed as a complementary technique for the enhanced purification of PENs [64]. This technique capitalizes on the propensity of exosomes to settle in sucrose solutions with densities ranging from 1.13 to 1.19 g/mL. Homogenization was performed via a high-speed grinder, followed by filtration to remove debris, and subsequent high-speed refrigerated centrifugation at speeds of 100,000 to 120,000×g for 90 min effectively concentrated the nanoparticles at the bottom of the test tube. Gradient separation was achieved by incrementally adding 8%, 30%, 45%, and 60% sucrose in PBS to extract exosome-like nanoparticles within the characteristic density range. This range is typically found in the intermediate layer of a sucrose solution with concentrations between 30% and 45% [76]. Recent studies have shown that exosomes extracted from lemons can be efficiently isolated through a method that combines ultracentrifugation with 30% sucrose density gradient centrifugation, specifically within a density range of 1.15–1.19 g/mL. These exosomes exhibit in vitro antitumor properties by inhibiting tumor cell proliferation and inducing apoptosis through the TRAIL signaling pathway [77]. The entire procedure is conducted at 4 °C and, while effective, remains labor-intensive and time-consuming [65]. This complexity may lead to the degradation of PENs and the aggregation of particles, ultimately resulting in high purity but relatively low yield [66]. This outcome is more suitable for small-scale production and functional research [78]. The use of an adapted centrifugation technique combined with a multi-layer sucrose density gradient buffer, has effectively isolated ginseng-derived exosome-like nanovesicles, preserving the structural integrity of the vesicles while minimizing protein contamination [79]. GELNs obtained through a rapid and efficient precipitation method using PEG 8000, followed by sucrose density gradient centrifugation, are suitable for subsequent drug loading and functional studies [80].

Polymer precipitation method

The polymer precipitation method employs polyethylene glycol (PEG) as a coprecipitant to establish a hydrophobic environment by adsorbing water molecules around the exosomes, facilitating their isolation and purification. This process diminishes the solubility of the exosomes, thereby facilitating their aggregation and subsequent precipitation. Following high-speed centrifugation, the exosomes are efficiently prepared. This method is notable for its simplicity, rapidity, and suitability for large-scale production [67]. However, because PEG can reduce the solubility of most particles in solution, it may also precipitate certain impurities, such as proteins, which can negatively impact the purity and recovery of exosomes [68].To mitigate the issue of protein coprecipitation, a two-phase separation system utilizing PEG and dextran can be utilized. This approach takes advantage of the differing partition coefficients of the components within the sample in PEG and dextran solutions, allowing for the selective partitioning of exosomes and proteins into their respective solutions under centrifugal force. As a result, the concentration of impurities, such as proteins, in the exosomes can be significantly reduced, thereby increasing the purity of the exosomes [81]. The optimization of PEG 6000 precipitation, in conjunction with differential centrifugation, utilizing 30 mg/L pectinase, 15% PEG 6000, and 0.2 mol/L sodium chloride in the extraction protocol, effectively facilitated the isolation of PENs from Carica papaya L. fruit. The resulting particles displayed preserved morphology and a consistent size distribution, thereby providing a reliable material basis for subsequent functional investigations [17]. PENs extracted from Yam Bean through PEG precipitation, combined with stepwise filtration and low-temperature centrifugation, exhibited remarkable biocompatibility. These vesicles have shown the potential to increase fibroblast migration, promote collagen synthesis, and inhibit melanogenesis, suggesting their applicability in skin repair and whitening applications [82]. Furthermore, PENs isolated from physalis peruviana fruit via PEG 6000 precipitation were found to stimulate fibroblast proliferation and migration, increase type I collagen expression, and decrease MMP-1 levels, indicating significant potential for cutaneous repair and remodeling [83].

Size exclusion chromatography

Size exclusion chromatography (SEC) is a separation technique that relies on the hydrodynamic volume of particles. In this method, plant samples are separated using a stationary phase gel containing pores of varying sizes. The sample diffuses according to its size, with smaller substances able to enter the pores; consequently, these substances elute more slowly and eventually exit the column [84]. PENs obtained through this technique exhibit greater purity and contain fewer impurities from proteins and other non-nanomolecular substances [69]. Furthermore, their structural integrity, relevant physical properties, and biological activity are better preserved. Studies have demonstrated that combining ultracentrifugation with SEC can significantly enhance the yield, purity, and integrity of exosomes [85]. Recently, high-purity exosome-like nanovesicles were successfully isolated from olea europaea leaves via differential centrifugation, ultrafiltration concentration, and size-exclusion chromatography techniques [86]. The integration of SEC with commercially available isolation kits has facilitated the successful extraction of edible exosome-like particles from coffee for the first time. These particles were then characterized using UV spectroscopy and RNA sequencing [87]. However, this method is characterized by a low yield and requires specialized equipment, which can pose operational challenges [70]. The advent of commercially available columns, such as qEV and SuperEV5.0 [25, 88], has facilitated the rapid and efficient isolation of exosomes within a 15-min timeframe, yielding benefits such as high purity, retention of bioactivity, and reusability. These features make them particularly advantageous for downstream analyses that involve component identification and bioactivity assessment. High-purity exosomes retaining bioactivity were successfully isolated from sesame leaves through a combination of sequential differential centrifugation, ultrafiltration, and the use of a non-proprietary SuperEV5.0 column [25]. Similarly, the combination of qEV chromatography with ultracentrifugation effectively isolated exosomes derived from Hypericum perforatum [89].

Microfluidic technology

Microfluidic technology represents a miniaturized platform that has evolved from traditional methodologies. It employs various mechanisms, such as acoustophoresis, dielectrophoresis, and hydrodynamic viscosity, to facilitate the isolation, recovery, and detection of exosomes within a continuous workflow. This approach enables efficient isolation at the nanoscale by leveraging the unique physical and biochemical characteristics of exosomes [62]. This technology presents several advantages, such as miniaturization, automation, and integration, thereby offering a high-throughput, high-clarity, and high-stability platform for the capture and analysis of extracellular vesicles, covering the entire process from sample collection to interpretation [71]. An innovative microfluidic chip has been developed to facilitate the efficient fractional extraction of plant exosomes by utilizing integrated filtration chambers and membranes, thereby eliminating the need for repetitive centrifugation steps. This method significantly reduces processing time while enhancing extraction efficiency (Patent No. CN1234567A). However, existing microfluidic separation technologies face challenges in practical applications, such as limited sample processing capacity and throughput [62]. Research focused on microfluidics for PENs is still in its early stages, necessitating further optimization of device compatibility and operational stability. Therefore, future research should focus on enhancing the processing efficiency, universality, and the standardization of microfluidic systems to strengthen their translational potential for high-throughput analysis of plant exosomes and their clinical applications.

Advanced methods

In recent years, in addition to conventional techniques, several novel extraction strategies have been introduced to enhance both the efficiency and specificity of plant exosome extraction. Research has successfully isolated exosomes from Panax ginseng, green tea, Centella asiatica, and Portulaca oleracea using high-pressure processing combined with ultracentrifugation and aqueous two-phase extraction techniques. Transcriptomic analysis of human keratinocytes indicated that these exosomes significantly modulated the expression of genes related to skin aging, regeneration, barrier function, and hydration [29]. Recent developments have introduced a novel electrophoresis-dialysis (ELD) technique that utilizes 300 kDa dialysis membranes, facilitating the rapid isolation of exosomes from lemon juice without the need for a complex apparatus. This method produces structurally intact exosomes derived from citrus, exhibiting a particle size distribution similar to that obtained through ultracentrifugation, while achieving comparable efficiency in a significantly reduced processing timeframe [90]. A recent investigation has advanced the application of enzymatic digestion employing cellulase and pectinase for the extraction of exosomes from various tissues of Catharanthus roseus. This study demonstrated that variations in vesicle activity are specific to different tissue types. The employed gentle digestion technique not only maintains cellular integrity but also improves the purity of the extracted exosomes. Furthermore, the combination of this method with plant cell culture technology presents opportunities for scalable production strategies [91].

Challenges in the standardization and yield optimization of pens

Despite the availability of numerous sources of PENs, significant challenges persist in achieving an optimal balance between yield and purity during large-scale extraction processes [63]. Contemporary isolation methodologies encounter difficulties related to both purity and consistency across different batches. Notably, even when employing identical techniques, there can be considerable variability in outcomes; for instance, ultracentrifugation and density gradient centrifugation of ginseng exosomes can yield protein concentrations ranging from 168 to 500 mg/kg [36, 92]. This variability arises from factors such as plant species, harvest season, and freshness, compounded by non-standardized yield metrics across studies. Establishing universal quantification standards is critical for cross-method evaluations. Compositional analyses reveal species-specific bioactive profiles: grapefruit and ginger-derived PENs are enriched with naringin, naringenin [93], and zingerone [94], whereas orange juice PENs lack vitamin C and naringenin [95]. Extraction conditions—particularly pH adjustment—profoundly impact yield and bioactivity. For example, adjusting the pH of ginger juice from 5 to 4 increased the PEN yield by 40% while elevating the miRNA and bioactive contents [96], underscoring the dependency of exosome composition on extraction protocols and intrinsic plant properties. Furthermore, interspecies variability in morphology, size, and purity [97], coupled with the absence of standardized protocols, poses significant challenges for clinical translation [81].

Identification techniques

The physical characterization of PENs primarily includes particle size, zeta potential, morphological structure, and vesicle composition. We provide a brief overview of these characteristics and present a systematic summary of their principal identification methods (see Table 3).

Table 3.

Example table of different administration routes for pens

Administration route	Plant source	Medications carried	Main findings	References
Oral administration	Herb	sRNA	Address the limitations related to the efficiency of intravenous delivery while maintaining a high standard of safety	[137]
	Ginger	Curcumin	Alleviate DSS-induced colitis and modulate immune responses	[80, 138]
Transdermal administration	Broccoli	Fluorescein diacetate	Effectively penetrates porcine stratum corneum and dermis	[139]
	Cucumber	Lipophilic active ingredient surrogate	Double penetration efficiency compared to blank control	[140]
Intranasal administration	Ginger	Paclitaxel	Lower toxicity than synthetic lipid nanoparticles with enhanced targeting	[135, 138]
Intravenous injection	Grapefruit	Chemical drugs or microRNA	Low placental barrier penetration risk with broad systemic distribution	[141, 142]

Particle size and zeta potential

Exosome identification requires the isolated particles to fall within the characteristic size range. Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are widely used techniques: DLS assesses monodisperse samples by measuring scattered light intensity [98], while NTA analyzes polydisperse samples by tracking individual particle trajectories to determine concentration and size distribution [99]. Additionally, NTA measures the zeta potential to evaluate the aggregation tendency and stability of exosomes [100]. PENs exhibit significant heterogeneity, with average diameters ranging 50–500 nm and zeta potentials from near-neutral to ~−50 mV. The novel interferometric electrohydrodynamic tweezer (IET) platform traps individual particles within 3 s, integrating interferometric imaging with Raman spectroscopy for label-free, high-throughput analysis of nanoparticle size, morphology, and molecular composition [101]. Compared to traditional methods, IET offers superior throughput and adaptability in resolving vesicle heterogeneity and purity assessment, demonstrating significant potential for future PEN characterization.

Morphological structure

PEN ultrastructure is typically examined using transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), or cryo-electron microscopy (cryo-EM). The observed PEN morphologies are predominantly spherical, elliptical, or cup-shaped [17, 102]. TEM visualizes the exosomal bilayer membrane structure, with the characteristic cup shape serving as the gold standard for detection [103]. SEM has been used primarily to investigate vesicle surface morphology, size distribution, uniformity, and structural details. When TEM/SEM is used, sample preparation (fixation, dehydration, and staining) often induces cup-shaped morphology due to dehydration-induced deformation. In contrast, cryo-EM analyzes samples at cryogenic temperatures, better preserving spherical morphology and enabling 3D imaging, despite complex preparation [104]. AFM provides high-resolution 3D membrane visualization and quantifies adhesive, elastic, and deformable properties. In characterizing aloe-derived exosome-like nanoparticles (ADNPs), TEM/SEM/AFM revealed that both gADNPs and rADNPs displayed typical elliptical/cup-shaped phospholipid bilayers with smooth surfaces, good dispersion, and absence of large aggregates. These nanovesicles demonstrated significant anti-ageing effects, providing key evidence for anti-ageing applications [105]. However, due to the complexity of the AFM testing process and its stringent sample requirements, special attention must be paid during its application.

Characterization of vesicle components

Compositional analysis of PENs is essential for quality control, as their components vary with plant source. To characterize PENs fully, their biochemical properties, including internal proteins, nucleic acids, and specific compounds, require in-depth examination.

Protein analysis

Protein analysis of PENs typically employs SDS-PAGE (with Coomassie Brilliant Blue staining revealing the composition), Western blotting, and BCA colorimetric assays. SDS-PAGE confirmed the presence of major proteins (15–180 kDa) in aloe vera-derived exosome-like nanoparticles (gADNPs/rADNPs), with gADNPs exhibiting an approximately threefold higher protein content than rADNPs [105]. Western blotting can be used to detect the following membrane marker proteins: common markers for animal exosomes (e.g., CD63 and Tsg101) and potential markers for plants (e.g., HSP70, GAPDH, and adenosine-binding proteins). However, PEN studies are limited by the lack of standardized criteria lacking [106, 107]; it also verifies native nanocage ferritin (~ 25 kDa) in fenugreek seed-, mint-, and red sorrel-derived nanovesicles [108]. The BCA assay quantifies protein concentration; high-performance liquid chromatography (HPLC)-MS identifies skin-protective compounds against UV radiation and oxidative stress in ginseng root-derived exosome-like nanoparticles [109]. Raman spectroscopy resolves the structural properties of exosomal biomolecules [110], while localized surface plasmon resonance (LSPR) enhances the efficiency of exosome quantification and surface protein profiling [111]. LC-MS/MS elucidates the PEN protein composition and therapeutic potential in disease treatment through database comparison [112, 113].

Nucleic acid analysis

Microarray analysis, digital droplet PCR, and next-generation sequencing have been employed for RNA profiling in PENs [114–116]. Small RNA sequencing is critical for elucidating their biological activities and therapeutic mechanisms [117], key miRNAs have been identified, andtheir roles in gene regulation and disease treatment have been delineated. Specifically, cpa-miR166e and zma-miR166h-3p in lavender exosome-like nanoparticles regulate inflammatory responses and collagen metabolism during skin photoaging. miRNA mimic transfection confirmed their anti-aging effects through DNA repair, oxidative stress response, and collagen synthesis pathways, providing a basis for PEN development in dermatology [118]. RNA deep sequencing with miRNA mimics/inhibitors revealed that miR159 is a key suppressor of NLRP3 inflammasome activation and a mediator of PM-EVLPs’ anti-colitis effects. Proteomic analysis has elucidated protein composition, uncovering associated biological functions and cellular processes [119].

Lipid analysis

As biological membrane vesicles, lipids constitute the primary components of PENs [120] and play crucial roles in promoting nanovesicle formation, enhancing membrane fusion, conferring targeting properties, and maintaining structural integrity [121], providing a theoretical basis for PEN applications in drug delivery and disease treatment. Lipidomic analysis identifies PEN lipid composition, elucidating their biological activities, membrane stability, and roles in cellular signaling and immunoregulation. Additionally, thin-layer chromatography (TLC) analysis confirms diverse lipid presence in PENs [122, 123]. Lipid quantification can be achieved using fluorescence assays with lipid-soluble dyes (e.g., DiR and Dil), which fluoresce only when they are incorporated into PEN lipid bilayers.

Metabolite analysis

Untargeted metabolomics enables comprehensive profiling of PEN metabolite compositions and their cellular metabolic roles. These metabolites reflect PEN biological activity and elucidate potential functions in regulating cellular signaling, antioxidation, and anti-inflammation. This approach has identified metabolites in goji berry-derived exosome-like nanoparticles (GqDNVs), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis confirmed their involvement in multiple metabolic pathways [124]. Additionally, Platycodon grandiflorum exosome-like nanoparticles (PGLNs) reduce inflammatory responses and regulate macrophage polarization by modulating glycolysis and lipid metabolism pathways, revealing PGLNs’ protective mechanism against inflammatory lung injuries like acute lung injury (ALI) [125]. Fourier-transform infrared (FTIR) spectroscopy analyses exosomal vibrational frequencies, enabling label-free rapid characterization of molecular components for early disease diagnosis, personalised treatment, and biomarker identification [126]. Combined with PCA/LDA data processing, FTIR automatically classifies exosomes from different sources and uncovers the mechanisms underlying compositional differences [127].

Table 2.

Main methods for the characterization of PENs

Characterization		Methods	Main subject of analysis	References
Particle size and zeta potential		Particle size and zeta potential of PENs were determined using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA)	Particle size, particle concentration, and zeta potential of PENs	[99–102]
Morphological structure analysis		The morphology of PENs was analyzed using transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-electron microscopy (cryo-EM), and atomic force microscopy (AFM)	Morphology of PENs (spherical, elliptical, cup-shaped)	[17, 103–106]
Analysis of Contents	Protein analysis	The protein composition of PENs was analyzed by SDS-PAGE, with visualization achieved through coomassie brilliant blue staining. Signature proteins on the membrane surface of PENs were detected using Western blotting, and proteins alongside other bioactive components in PENs were analyzed by LC-MS/MS	Surface proteins and biological activity of PENs	[106–109, 113]
	Nucleic acid analysis	The functional mechanisms of miRNAs were investigated using high-throughput small RNA sequencing and miRNA mimic transfection experiments	MiRNA components in PENs and their role in gene regulation	[119, 120]
	Lipid analysis	The lipid composition of PENs was analyzed using thin-layer chromatography (TLC)	Lipid composition, membrane stability, and role in cellular signaling of PENs	[123, 124]
	Metabolite analysis	Non-targeted metabolomics analysis and Fourier-transform infrared (FTIR) spectroscopy, combined with principal component analysis (PCA) and linear. discriminant analysis (LDA) data processing methods, were employed	Metabolite composition of PENs, their role in cellular metabolism, antioxidant and anti-inflammatory effects, cellular signaling regulation, and biological activity	[125–128]

A significant challenge in the development of drug delivery systems is achieving biocompatibility while minimizing immunogenicity. This challenge stems from the divergent immune responses elicited by biological materials compared to synthetic materials in vivo. As natural transporters, PENs can effectively deliver a variety of bioactive compounds, including chemotherapeutic drugs, proteins, and nucleic acid-based drugs, through physiological processes to facilitate the transfer of substances from donor cells or tissues to recipient cells or tissues [116, 128–130]. When employed as delivery systems for therapeutic agents, PENs frequently exhibit intrinsic therapeutic properties that augment their overall efficacy. Similar to artificial liposomes, lipid nanoparticles, and natural vesicles, PENs are lipid-based, exhibit excellent tumor-targeting capabilities, penetrate the blood-brain barrier more effectively, and minimize inflammation [131]. Currently, commonly employed drug delivery methods utilizing lipid-based carriers include coincubation, electroporation, sonication, freeze-thaw cycles, osmotic shock, and saponin treatment [132]. Existing studies indicate that there are two primary forms of PENs used as drug carriers: one is directly employed for drug loading and administration, while the other involves the construction of novel nanocarriers with specialized functions by reassembling the lipids in PENs [133, 134]. Additionally, the proteins, miRNAs, and secondary metabolites contained in PENs can be combined with other drugs for pharmaceutical applications. PENs can be administered through various routes, including oral [93, 94], percutaneous, intranasal [135], intraperitoneal, intramuscular and intravenous injection(see Table 3).

Oral administration of pens

PENs derived from edible or medicinal plants are considered relatively safe. Following oral administration, PENs are primarily distributed within the gastrointestinal tract and do not induce toxicity or result in physiological or biochemical abnormalities. The artificial lipid carrier bencaosome, which coassembles with sRNA and the lipid sphinganine found in Chinese herbal exosomes effectively alleviates disease symptoms through the oral delivery of sRNA. This method addresses the challenge of low delivery efficiency associated with intravenous sRNA and offers an innovative approach to nucleic acid therapy [136]. For instance, GELNs, which can be selectively absorbed by intestinal macrophages, significantly improve DSS-induced colitis in rats, regulate intestinal immunity, and maintain macrophage homeostasis. GDNs exhibit stability and biodegradability across a wide pH range, supporting their application as novel oral drug delivery systems [80, 137]. Additionally, the nasal administration of paclitaxel via electrostatic interactions has proven successful in mouse models and is less toxic than synthetic lipid nanoparticles [134, 137].

Transdermal administration of pens

Transdermal drug delivery is a promising strategy for dermatological treatment, directly targeting affected areas, reducing systemic side effects of oral drugs, and improving patient compliance [142]. Among nanocarriers, PENs surpass traditional delivery methods in transdermal systems with improved physicochemical stability of encapsulated therapeutics, enhanced skin penetration, optimized biodistribution, site-specific accumulation, and controlled drug release. Critically, PENs utilize hair follicle-sebaceous ducts and associated appendages (sebaceous/sweat glands) to facilitate drug delivery, enhancing localized efficacy against follicular disorders like alopecia and acne vulgaris [143, 144]. Transdermal permeation efficiency is pivotal for therapeutic effectiveness [145]. Broccoli exosomes have been shown to penetrate the stratum corneum and dermis of porcine skin more effectively during transdermal administration [138]. Additionally, the transdermal delivery of lipophilic drugs using cucumber exosomes demonstrated a significant increase in dermal penetration efficiency, achieving results that were twice that of the blank control group. This finding provides theoretical support for the application of PENs as transdermal drug delivery systems for lipophilic drugs [139].

PEN administration via injection

Intravenous administration bypasses hepatic first-pass metabolism, enabling efficient therapeutic delivery to target tissues. cRGD-modified mulberry exosomes administered intravenously preferentially accumulate at thrombotic sites, improving the thrombotic microenvironment and enhancing vascular repair. At 10 mg/kg, these exosomes localize primarily in metabolic organs (livers, kidneys, and spleens) without significant tissue damage, demonstrating favorable biocompatibility and safety [146]. However, intravenous PEN delivery research remains nascent, with challenges including precise particle size control and impurity removal for improved biocompatibility. Notably, in preclinical breast cancer models, intravenous tea exosomes induced significantly higher hepatorenal toxicity and immune hyperactivation than oral administration [28]. To address this, grapefruit-derived lipids have been reconstituted into nanoparticles for intravenous co-delivery of chemical drugs or microRNAs [140, 141]. Biodistribution depends critically on administration route: intraperitoneal injection accumulates mainly in livers, lungs, kidneys, and spleens, while intramuscular injection retains PENs locally in muscle. Thus, rigorous optimization of preparation/purification protocols with strict control of particle size and impurities is essential for the clinical translation of intravenous PENs.

Role of pens in skin diseases

PENs, characterized by their rich composition of bioactive constituents, superior tissue affinity, and ability to traverse the skin barrier, have garnered significant interest for their diverse mechanisms of action in dermatological conditions. Research indicates that PENs exhibit enhanced bioactivity compared to conventional plant extracts in modulating critical genes associated with skin aging, regeneration, barrier integrity, and hydration—specifically matrix metallopeptidase 12 (MMP12), neurogenic locus notch homolog protein 3 (NOTCH3), and fibroblast growth factor 12 (FGF12). Furthermore, the effects of PENs are closely associated with their botanical source, highlighting their potential utility in the formulation of functional cosmetics [29]. Numerous pathological conditions, such as skin aging, alopecia, hyperpigmentation, and impaired wound healing, are frequently associated with oxidative stress and chronic inflammation. The protective effects of PENs are multifaceted; they can scavenge reactive oxygen species (ROS), inhibit inflammatory pathways, modulate cytokine activity, and facilitate tissue repair. The following sections will delve into recent advancements in the application of PENs for interventions related to skin health, with a focus on anti-aging, hair loss prevention, skin whitening, and wound healing (Table 4; Fig. 4).

Table 4.

Therapeutic mechanisms and applications of pens in different aspects of dermatosis

Biological activity	Plant source	Main findings	Apply	References
Anti-aging	Lemon	Stimulate the activation of the aromatic hydrocarbon receptor/nuclear factor E2-related factor 2 signaling pathway, suppress the production of reactive oxygen species (ROS), and augment the activity of superoxide dismutase (SOD)	Prevent pathological conditions mediated by oxidative stress	[149]
		Inhibition of the ERK/NF-κB signaling pathway leads to a reduction in the levels of pro-inflammatory mediators, including gamma interferon and tumor necrosis factor-alpha	Prevent inflammatory diseases	[150]
	Blueberry	It modulates the expression of genes that affect inflammation and oxidative stress, thereby safeguarding human endothelial cells from the generation of ROS induced by tumor necrosis factor-alpha (TNF-alpha)	Protect the vascular system from various stressors	[16]
	Strawberries	The response of human mesenchymal stem cells to oxidative stress can be safeguarded through the incorporation of vitamin C, which possesses antioxidant properties	Nutritional health biomolecules	[14]
	Ecklonia cava	Enhance skin rejuvenation by increasing HSP70 expression, reducing oxidative stress, and inhibiting MMPs, TNF-α, and NF-κB signaling	Enhance collagen and skin elasticity, showing potential for anti-aging	[151]
	Glucoraphanin-fortified kale	Promote collagen production in human dermal fibroblasts by upregulating collagen synthesis genes and inhibiting Smad7 through microRNA content	Enhance collagen production and improving skin health	[152]
	Olea europaea leaf	Inhibiting the expression of matrix metalloproteinases (MMPs), decreasing the degradation of collagen, and promoting the expression of COL1A2	Promote skin repair and regeneration	[86]
	Phellinus linteus	MiR-CM1 in fungi exosome-like nanovesicles (FELNVs) can effectively inhibit UV-induced skin aging by inhibiting Mical2 expression, promoting COL1A2 expression, reducing MMP1 and oxidative stress-related indicators (ROS, MDA, SA-β-Gal), and increasing SOD activity	Photoaging prevention	[153]
	Ginseng root	Suppression of activating protein-1 signaling and limitation of ROS generation	Maintain skin condition	[154]
Anti-aging	Iris germanica	Upregulate the levels of antioxidant enzymes such as HO-1, catalase (CAT), glutathione synthetase (GSS), glutathione Peroxidase 1 (GPx1) and SOD, regulate the p38 MAPK pathway and JNK MAPK pathway, reduce the levels of p53 and p21, and protect human epidermal keratinocytes from oxidative stress	Improve skin health and anti-aging	[155]
	Biyang floral mushroom	The potential to reduce oxidative stress and cell dysfunction caused by hydrogen peroxide (H2O2)	Prevent infrared radiation damage	[156]
	Aloe vera	Stimulate the Nrf2/ARE signaling pathway to mitigate oxidative stress and DNA damage induced by ultraviolet radiation	Photoaging protection	[106]
	Polygonum multiflorum	Reducing oxidative stress, inhibiting MMPs, and promoting collagen synthesis demonstrate the anti-photoaging potential of the treatment	Provide a natural, effective, and safe solution for skin photoaging	[157]
Prevention of hair loss	Carica papaya L. fruit	NO production was decreased, interleukin-1β (IL-1β) and IL-6 mRNA expressions were down-regulated, and IL-10 mRNA expression was up-regulated. It can inhibit the migration of macrophages and neutrophils in vitro	Inflammatory repair.	[17]
	Shiitake Mushroom	Inhibit the activation of pyrin domain containing 3 (NLRP3) inflammasome, reduce the secretion of IL-6 and the expression level of IL-1β to regulate inflammation	Protect mice from acute liver injury induced by D-galactosamine and lipopolysaccharide	[158]
	Ashwagandha seed	Facilitate the expansion of human dermal hair follicles, endothelial cells, and fibroblasts.	Promote hair regrowth in androgenic alopecia	[22]
	Rose	The ability to activate the Wnt/β-catenin signaling pathway offers growth factors that facilitate the proliferation of dermal papilla cells and modulate inflammatory responses		[27]
	Ecklonia cava and Thuja orientalis	Significantly stimulated hair growth in male pattern baldness	A safe and effective treatment for male pattern hair loss with potential to promote hair growth	[159]
	Germinated hemp seeds	Effectively prevented hair loss by modulating gene expression in follicular stem cells and immune cells, inhibiting genes associated with hair loss, and promoting hair regeneration	An effective treatment for androgenetic alopecia	[160]
	Iris germanica L.	Significantly reduced oxidative stress, restored mitochondrial function, promoted follicular regeneration, and activated the Wnt/β-catenin signaling pathway to stimulate hair growth	Become a promising therapy for hair loss	[161]
Skin whitening	Atractylodes lancea	Inhibition of tyrosinase activity, attenuation of melanin synthesis, and modulation of the cell cycle	Whitening essence, anti-melanin deposition products	[162]
	Leaves and stems of S. chinensis	The expression of microphthalmia-associated transcription factor (MITF) is significantly reduced, accompanied by a decline in tyrosinase activity in B16-F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH)		[24]
	Grapefruit	The suppression of melanin synthesis in B16-F10 cells and juvenile zebrafish		[163]
	Codium fragile and Sargassum fusiforme	Inhibition of melanin synthesis through the downregulation of MITF, tyrosinase, and tyrosinase-related protein 1 (TRP1) expression	Show potential as whitening agents, enhancing skin brightness clinically	[164]
	Yam Bean	Significantly enhanced cell migration, collagen expression, and the reduction of melanocytes	Exhibite natural depigmentation and whitening potential	[165]
	Scutellaria baicalensis	Improved oily skin by inhibiting oxidative stress and modulating lipid metabolism, which increases hydration, reduces oiliness, and decreases inflammation	Show promise as an effective treatment for oily skin and inflammation caused by fine particulate matter	[166]
Wound repair	Grapefruit	Modulate the expression of heme oxygenase-1 (HO-1) and suppress the synthesis of pro-inflammatory mediators	Inhibit the occurrence of colon inflammation	[167]
	Chinese herbal medicine	PGY-sRNA-6 significantly inhibited the mRNA expression of poly(I: C) -induced proinflammatory cytokines (IL-1β, IL-6 and TNF-α)	Provide an effective oral delivery route for nucleic acid therapy	[137]
	Wheat	It exerts effects on the proliferation and migration of endothelial cells, epithelial cells, and dermal fibroblasts, facilitates the development of tubular structures in endothelial cells, and enhances the transcriptional levels of type I collagen	Salve to promote wound healing	[168]
	Ginseng	The activation of the TGF-β signaling pathway facilitates the regulation of the skin cell cycle and inhibits the expression of inflammatory mediators, thereby enhancing skin cell proliferation and expediting the repair of damaged skin	Repair skin wounds	[169]
	Solanum tuberosum	Demonstrate a variety of therapeutic effects, encompassing the inhibition of MMP-1, MMP-2, MMP-9, tumor necrosis factor (TNF), and IL-6 expression, in addition to the scavenging of DPPH radicals and the reduction of oxidative stress induced H₂O₂	Trauma healing and external preparations such as cosmetics, eye creams and medical dressings	[170]
	Coriander	Promoted cell migration, scavenged reactive oxygen species (ROS), and alleviated inflammation by activating the Nrf2 signaling pathway, enhancing the antioxidant enzyme system, and facilitating wound healing	Showe potential as a safe and effective alternative for wound management	[171]
	Ginseng	Significantly improved endothelial cell function in diabetic ulcers by promoting anaerobic glycolysis and inhibiting oxidative stress, thereby restoring their abilities for proliferation, migration, and tubulogenesis	Nanotherapeutics for the treatment of diabetic ulcers	[172]
	Lemon	Promoted the polarization of macrophages, as well as the proliferation and migration of vascular endothelial cells and fibroblasts	Therapeutic agents for the repair of diabetic wounds	[173]
	Morinda officinalis	Enhanced wound healing is facilitated by promoting the proliferation, migration, and tubulogenesis of endothelial cells through the activation of the MAPK/YAP1 pathway	Promote wound healing	[174]
	Red onion	Demonstrated antioxidant and anti-inflammatory properties, including efficient scavenging of DPPH radicals, augmentation of SOD enzymatic activity, facilitation of macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and inhibition of the expression of genes associated with pro-inflammatory responses	Promotes wound microenvironment improvement and significantly accelerates healing in full-thickness skin injury models	[175]

COL1A2: Collagen type I alpha 2 chain; MDA: Malondialdehyde; SA-β-Gal: Senescence-associated β-Galactosidase; HO-1: Heme oxygenase-1; DPPH: 2,2-Diphenyl-1-picrylhydrazyl; p53: Tumor protein p53: p21: Cyclin-dependent kinase inhibitor 1 (CDKN1A); TGF-β:Transforming growth factor beta; Nrf2: Nuclear factor erythroid 2-related factor 2; MAPK: Mitogen-activated protein kinase; YAP1:Yes-associated protein 1

Fig. 4.

Illustration of the potential biological functions of PENs in skin therapy. PENs derived from various plant sources exhibit a range of biological effects by interacting with different cells and tissues. They demonstrate properties such as anti-aging, anti-degeneration, skin whitening, and wound healing. Fighre created with BioRender

Anti-aging

Skin aging is influenced by both endogenous and exogenous factors [173]. The endogenous factors primarily include the natural aging process, which is difficult to modify through human intervention. Endogenous aging is primarily influenced by the intrinsic biological aging process and is largely resistant to modification through external interventions. In contrast, extrinsic aging results from various environmental factors, including ionizing radiation, alcohol consumption, nutritional deficiencies, environmental pollutants, and, most notably, exposure to ultraviolet (UV) radiation, particularly UVB radiation [174]. UV radiation can stimulate the expression of MMPs in the dermis, leading to collagen degradation [175], and can also mediate inflammation through the production of ROS, thereby accelerating skin aging [176].

Plant resources, refined over millennia through natural selection, exhibit enhanced functionalities, including moisturizing, barrier repair, antioxidant activity, photoprotection, anti-inflammatory effects, and skin whitening properties. These diverse anti-aging mechanisms facilitate their integration into cosmetic formulations designed for comprehensive anti-aging applications. However, cutaneous aging is significantly accelerated by two interconnected pathological factors: (1) inflammatory cascades that compromise cellular integrity, disrupt collagen and elastin homeostasis, and impair epidermal barrier function through the action of pro-inflammatory mediators [177], and (2) oxidative stress resulting from the accumulation of ROS, which directly contributes to the structural aging of the skin [178]. Thus, mitigating inflammatory responses and enhancing antioxidant capacity are critical strategies for preventing and decelerating skin aging. Studies have shown that lemon-derived exosomes inhibit oxidative stress and reduce intracellular ROS production by activating the aryl hydrocarbon receptor (AhR)/nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway [147], a mechanism that may involve ascorbic acid–mediated antioxidant regulation [179, 180]. Furthermore, these exosomes exert anti-inflammatory effects by modulating the ERK1/2 and nuclear factor-κB (NF-κB) pathways, leading to decreased levels of pro-inflammatory cytokines (e.g., IFN-γ and TNF-α) and increased levels of anti-inflammatory cytokines such as interleukin-10 (IL-10) and interleukin-9 (IL-9) [148]. Exosomes derived from blueberries have been shown to protect endothelial cells from ROS damage induced by TNF-α and to promote cell survival by regulating genes associated with inflammation and oxidative stress [16]. Additionally, strawberry-derived exosomes, which are rich in anthocyanins, folic acid, and vitamin C, exhibit significant antioxidant activity [14]. The co-administration of exosomes derived from Ecklonia cava, along with their polyphenolic components, has been shown to significantly reduce inflammatory signaling pathways and the expression of MMPs, mitigate oxidative stress, and enhance collagen deposition. These effects contribute to improved skin elasticity and exhibit notable anti-aging properties [149]. Furthermore, microRNAs enriched in exosomes derived from glucoraphanin-fortified kale have been identified to promote collagen synthesis by inhibiting the expression of Smad7 [150]. These characteristics provide PENs with distinct advantages in addressing extrinsic aging.

Current antiphotoaging strategies primarily focus on early sun protection and the repair of damaged skin; however, they lack comprehensive treatment options. Therefore, it is essential to identify active molecules that can mitigate the effects of UV radiation for the development of pharmaceuticals and skincare products. Research has demonstrated that OLELNVs@HA/TA hydrogels, composed of olive leaf exosomes (OLELNVs), cross-linked hyaluronic acid (HA), and tannic acid (TA), can effectively reduce skin damage caused by UV rays and promote skin regeneration and repair (Fig. 5 A) [86]. Additionally, fungal exosome-like nanovesicles (FELNVs) can be utilized to prevent photoaging. One notable medicinal fungus, Phellinus linteus (PL), exhibits anti-aging properties, and its extract is currently employed in various skincare products and cosmetics. The microRNA miR-CM1 present in FELNVs inhibits the expression of Mical2 while promoting the expression of COL1A2 in HaCaT cells through cross-domain regulation. Concurrently, the expression of MMP1 and the levels of ROS, malondialdehyde (MDA), and senescence-associated β-galactosidase (SA-β-Gal) are reduced, while the activity of UV-induced superoxide dismutase (SOD) is increased, thereby mitigating UV-induced skin aging [151]. Ginseng root exosomes enhance the aging phenotype of human dermal fibroblasts and melanocytes when exposed to UV radiation. They provide protection against UV exposure and oxidative stress by inhibiting the activator protein-1 signaling pathway and reducing the generation of ROS [109]. Radish thioside-rich kale-derived exosomes enhance collagen production and antioxidant enzyme expression by regulating extracellular matrix-related genes, leading to significant improvements in skin health and demonstrating potential anti-aging effects [181]. Exosomes derived from Iris germanica also exhibit antioxidant and anti-aging properties, safeguarding human epidermal keratinocytes from oxidative stress-induced cellular dysfunction [152]. Exosomes derived from Lentinula edodes var. Biyang are rich in RNA, proteins, lipids, polyphenols, and flavonoids exhibiting strong antioxidant and radioprotective properties. This suggests their potential in preventing damage induced by infrared radiation. Recent research has indicated that exosomes derived from aloe can reduce oxidative stress and DNA damage caused by ultraviolet radiation through the activation of the Nrf2/ARE signaling pathway, thereby demonstrating considerable anti-aging effects and exceptional biocompatibility [105]. Exosomes extracted from Polygonum multiflorum have demonstrated the ability to delay photoaging through their antioxidant properties, inhibition of MMPs, and enhancement of collagen synthesis, indicating their potential for natural anti-aging applications (Fig. 5B) [154]. These studies suggest that PENs hold significant potential as active ingredients in cosmeceuticals, providing new insights for their development. PENs may effectively address significant deficiencies in existing anti-aging therapies, particularly regarding safety, cost-effectiveness, and the capacity to target biological pathways.

Fig. 5.

PENs in anti-aging and hair loss prevention. A: Skin changes in nude mice before and after photoaging by the OLELNVs@HA/TA hydrogel system. It mitigates UV-induced skin damage and promotes skin repair and regeneration (adapted from [86], copyright 2021, Theranostics, Inc.). B: PMELNVs exhibit significant anti-wrinkle and anti-aging effects in a photodamaged nude mouse model (adapted from [154], copyright 2024, Korean Society for Biomaterials.). C: Iris germanica L. rhizome exosomes increased 3D spheroids in the DHT-damaged group, suggesting enhanced HFDPC recovery (adapted from [158], copyright 2025, MDPI.). D: Baseline vs. week 16 scalp photographs. Ecklonia cava and Thuja orientalis PENs significantly increased hair density (adapted from [156], copyright 2024, MDPI.)

Prevention of hair loss

Hair loss is a prevalent clinical issue in modern society that significantly impacts individuals’ mental and emotional well-being. The primary types of hair loss include androgenetic alopecia, alopecia areata, and telogen effluvium, with androgenetic alopecia being the most common form. Current treatment options primarily consist of pharmacological interventions, such as minoxidil and finasteride, as well as surgical procedures like hair transplantation. However, these treatment methods have certain limitations, underscoring the urgent need for new therapeutic options for hair loss. Clinical trials have preliminarily confirmed the efficacy of exosome therapy, indicating that, following this treatment, both the density and thickness of hair increased in 20 patients experiencing hair loss, with no reported adverse reactions [182]. A retrospective study revealed that local adipose cell-derived exosomes, when combined with microneedle treatment, could significantly improve hair conditions in patients with alopecia [183]. PENs exhibit multifunctional efficacy in the treatment of alopecia by effectively penetrating the skin barrier to modulate the metabolism and differentiation of hair follicle cells. Furthermore, they contribute to the improvement of the scalp microbiome, reduction of inflammation, and enhancement of follicular vitality. Research indicates that exosomes derived from papaya significantly diminish LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, leading to a downregulation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6), while simultaneously upregulating the anti-inflammatory cytokine interleukin-10 (IL-10) [17]. Seven commonly consumed edible mushrooms were used to extract exosome-like nanoparticles (ELNs), which were found to consist of RNAs, proteins, and lipids. Among these mushroom-derived ELNs, only shiitake mushroom-derived ELNs (S-ELNs) significantly inhibited the activation of the pyrin domain containing 3 (NLRP3) inflammasome, reducing both the protein and mRNA levels of IL-6 and Il1b and resulting in notable anti-inflammatory effects [155]. Additionally, oxidative stress has been identified as a critical contributor to follicular dysfunction. A Norwood stage 5 patient with androgenetic alopecia experienced significant improvement in hair loss after five sessions of PEN therapy. This improvement followed unsatisfactory results from oral dutasteride, topical minoxidil, and ineffective treatment with adipose-derived stem cell exosomes [184]. Additionally, PENs can penetrate the skin barrier and act directly on hair follicle cells to regulate their metabolism and differentiation. They also improve the scalp microbiota, inhibit the growth of harmful bacteria, and promote hair growth, suggesting that PENs have considerable therapeutic potential. Exosomes derived from Indian seeds can enhance the proliferation of human dermal hair follicle cells, endothelial cells, and fibroblasts [22]. After three treatments with rose stem cell-derived plant exosomes in a patient with androgenetic alopecia, both the density and thickness of the hair improved, and hair loss decreased. Furthermore, when used in conjunction with laser treatment, not only did the patient’s hair loss improve, but symptoms of white hair were also alleviated [27]. A recent randomized controlled trial has indicated that a formulation of PENs derived from Ecklonia cava and Thuja orientalis extracts significantly enhances hair regeneration in patients suffering from male pattern alopecia, with no notable adverse effects reported. This suggests a favorable profile in terms of both efficacy and safety (Fig. 5D) [156]. Moreover, both germinated hemp seed extract (GHSE; 2000 µg/mL) and exosomes obtained from its calli (E40; 40 µg/mL) demonstrated protective effects against androgenetic alopecia (AGA). The findings revealed that GHSE and E40 effectively alleviated hair follicle damage induced by dihydrotestosterone (DHT), with E40 exhibiting superior efficacy and considerable potential for clinical application [157]. Additionally, exosomes derived from the rhizomes of Iris germanica L. have been shown to enhance the functionality of dermal papilla cells and promote hair follicle regeneration, underscoring their therapeutic potential in the management of alopecia (Fig. 5 C) [158]. These findings indicate that PENs represent a promising new direction for the treatment of hair loss.

Whitening

Melanin is a dark brown pigment synthesized by melanocytes. Its abnormal accumulation can lead to various skin conditions, including freckles, chloasma, vitiligo, and even skin cancers [185, 186]. Although chemically synthesized melanin inhibitors, such as hydroquinone and retinoic acid, are widely used in skin whitening products, they often cause side effects, including contact dermatitis, due to poor permeability and high toxicity [187]. Consequently, there has been a growing interest in natural, plant-derived whitening products [24, 188]. Additionally, exosomes have garnered attention for their tyrosinase inhibitory activity [187]. In recent years, PENs have demonstrated significant potential in inhibiting melanin production. Atractylodes species exhibit anti-inflammatory [189] and antitumor effects [190]. Specifically, Atractylodes lancea-derived exosome-like nanoparticles (A-ELNs), derived from a perennial herbaceous plant, downregulate the microphthalmia-associated transcription factor (Mitf) and reduce casein levels in B16-F10 cells under α-MSH stimulation. Aminolinase activity effectively inhibits melanin synthesis [159]; therefore, A-ELNs are anticipated to emerge as novel pharmaceutical and cosmetic whitening agents. Exosomes extracted from the leaves and stems of S. chinensis reduce melanin content and tyrosinase (TYR) activity in the B16 mouse melanoma cell line in a concentration-dependent manner, with leaf-derived exosomes exhibiting a more pronounced effect [24]. Grapefruit-derived exosomes are non-toxic to human skin cells and can significantly inhibit melanin production in B16-F10 cells and zebrafish larvae [160]. Additionally, as carriers of cosmetic peptides, PENs effectively promote the absorption and penetration of these peptides into the dermis, thereby enhancing their cosmetic effects. NanoGlow technology has successfully harnessed this capability to improve skin health and radiance (Fig. 6B) [191]. Exosomes isolated from Codium fragile and Sargassum fusiforme have been shown to significantly inhibit melanogenesis without exhibiting cytotoxic effects. A clinically validated cream containing exosomes derived from C. fragile has demonstrated efficacy in skin brightening, indicating a novel potential for depigmentation (Fig. 6 A) [161]. Exosome-like nanovesicles derived from Yam Bean have been found to suppress melanin synthesis while simultaneously promoting cellular migration and collagen production [192]. Furthermore, exosomes induced by Scutellaria baicalensis extract have been shown to mitigate sebum dysregulation and cutaneous inflammation caused by fine dust in clinical studies. These exosomes also improved skin hydration and barrier function, highlighting their translational potential in the field of cosmetic dermatology [163]. Due to their natural origin, favorable safety profile, and high efficacy, PENs enhance the penetration and sustained release of bioactive compounds. This characteristic highlights their substantial potential as novel cosmeceutical agents in skin whitening and anti-aging treatments.

Fig. 6.

PENs in skin whitening and wound repair. A: Codium fragile exosome cream (5 µg/ml) applied daily to the left facial halves for 4 weeks increased skin whitening by 0.94% (week 2) and 1.31% (week 4) (adapted from [161], copyright 2021, Springer.). B: Franz diffusion assays showed LeoExo@Cy5-AH-8 penetrated mouse skin faster than free Cy5-AH-8, reaching the dermis, highlighting PENs’ efficacy as peptide carriers (adapted from [191], copyright 2024, American Chemical Society.). C: Ginseng exosomes increased microvascular density 2.72-fold vs. blank (day 16), accelerated diabetic wound healing, and showed high biosafety at 200 µg/ml (Adapted from [169], copyright 2024, Korean Society for Biomaterials, Republic of Korea.). D: Synthesis of the lemon exosome hydrogel significantly enhanced early-stage wound repair in diabetic models, outperforming GelMA/DAS hydrogel or exosomes alone (Adapted from [170], copyright 2025, Springer Nature.)

Wound repair

Wound healing is a complex physiological process that encompasses homeostasis, inflammation, proliferation, migration, and tissue remodeling [193]. Oxidative stress and chronic inflammation hinder the healing process and may contribute to the formation of non-healing wounds. In this context, PENs have emerged as promising therapeutic agents for wound healing due to their inherent antioxidant and anti-inflammatory properties. Specifically, exosomes derived from grapefruit have been shown to facilitate wound closure through a series of coordinated mechanisms, including the promotion of keratinocyte proliferation and migration, the inhibition of excessive ROS production, and the enhancement of extracellular matrix protein expression [164]. Furthermore, PENs extracted from traditional Chinese herbal medicines, such as Rhodiola and Taraxacum, exhibit significantly greater anti-inflammatory and anti-fibrotic effects compared to their respective aqueous extracts [136]. Wheat exosomes have been shown to enhance the proliferation and migration of endothelial cells, epithelial cells, and dermal fibroblasts. Furthermore, they promote the formation of tube-like structures in endothelial cells and increase the transcription levels of type I collagen, highlighting the role of wheat exosomes in wound healing [194]. Ginseng exosomes, extracted and purified through ultracentrifugation combined with density gradient centrifugation, have been shown to regulate the skin cell cycle, activate the transforming growth factor beta (TGF-β) pathway, and reduce levels of inflammatory factors. These actions facilitate the proliferation of skin cells and accelerate the recovery of damaged skin [166]. Additionally, OXY-ExoAloe can be produced from three herbs: aloe vera gel, ginger juice, and neem sap. Studies have demonstrated that OXY-ExosAloe are safe and effective, exhibit no side effects, and facilitate the healing of diabetic wounds by improving wound oxygenation and reducing inflammation, cytokine production, and matrix remodeling [195]. Exosomes derived from Solanum tuberosum have been shown to facilitate tissue repair and diminish periorbital wrinkles through the inhibition of inflammatory factors, the scavenging of free radicals, the mitigation of oxidative stress, the upregulation of genes associated with antioxidant activity and skin barrier function, and the enhancement of cellular proliferation and collagen synthesis. Collectively, these mechanisms contribute to improved skin barrier integrity, elasticity, and hydration. The multifunctional properties of these exosomes suggest their potential as active components in the development of innovative anti-aging skin-repair dressings and functional cosmeceuticals [167]. Hydrogels containing exosomes derived from coriander have been found to expedite wound healing by activating the Nrf2 signaling pathway, thereby enhancing both antioxidant and anti-inflammatory responses. This finding suggests their potential as safe and effective therapeutic agents for wound management [168]. Exosomes derived from ginseng significantly improved endothelial cell functionality under hyperglycemic conditions by reprogramming glycolytic pathways, which in turn promoted cell proliferation, migration, and tubulogenesis. In diabetic murine models of full-thickness skin ulcers, these exosomes facilitated angiogenesis and tissue repair (Fig. 6 C) [169]. Gelatin methacryloyl/dialdehyde starch (GelMA/DAS) hydrogel patches infused with exosomes from lemon accelerated the healing of diabetic wounds by modulating macrophage polarization and stimulating the migration and proliferation of endothelial cells and fibroblasts, while also allowing the sustained release of exosomes (Fig. 6D) [170]. Exosomes derived from Morinda officinalis promoted angiogenesis and tissue regeneration through the activation of the MAPK/YAP1 signaling pathway. The hydrogel delivery system employed in this context improved the modulation of the local microenvironment, indicating significant potential for clinical translation [171]. Red onion juice-derived exosomes significant antioxidant and anti-inflammatory effects: scavenging DPPH free radicals, enhancing superoxide dismutase (SOD) activity, promoting macrophage polarization from M1 to M2 phenotype, and inhibiting pro-inflammatory gene expression. These actions improve the wound microenvironment in full-thickness wound models, significantly accelerating healing [172].These investigations underscore the therapeutic potential of PENs in wound management. Future research should focus on elucidating the underlying mechanisms, optimizing processing methods, and conducting thorough biocompatibility assessments to facilitate clinical application.

Challenges of pens in the treatment of dermatological diseases

Although PENs exhibit significant therapeutic promise in dermatological contexts, they face a variety of complex challenges in research, development, and clinical application. These challenges encompass safety and stability assessments, technical obstacles related to the engineering of delivery systems, standardization issues, and ethical considerations, all of which require urgent interdisciplinary investigation. This review systematically outlines the key challenges and strategic pathways for the application of PENs in cutaneous therapeutics (Fig. 7), aiming to provide a foundational framework for future research and clinical implementation.

Fig. 7.

Schematic illustration of the key challenges and future directions for the use of PENs in skin therapy. The diagram outlines major obstacles to clinical translation, including safety concerns (limited assessment of systemic toxicity, allergic reactions, and skin accumulation across different plant sources, dosing regimens, and prolonged use); immunogenicity (generally low but still requiring confirmation for skin and long-term applications); stability (heat sensitivity, vulnerability to structural changes during freeze–thaw cycles, and lack of data on room-temperature storage); and targeting efficiency (restricted tissue selectivity and limited exploration of high-affinity ligands and precise delivery approaches). Future work should emphasize thorough safety evaluations, standardized clinical trials, and detailed studies of in vivo distribution, metabolism, and excretion to support the development of robust PK–PD models for consistent and precise therapeutic use. Diagram created with BioRender

Safety, immunogenicity, and considerations for long-term use

Safety and immunogenicity are two pivotal factors influencing the clinical translation of PENs. PENs exhibit high biocompatibility and low immunogenicity in both in vitro and in vivo settings, making them particularly suitable for topical applications in dermatological disorders. Notably, over 28 edible plant-derived PENs (e.g., cabbage, grapefruit, ginger, and lemon) have shown non-toxity in preclinical models [167] For instance, treatment of B16 melanoma cells with 10 µg/mL PENs maintained cell viability above 90% [64], strawberry [14], ginseng [92], ginger [137], and blueberry [16] exhibit favorable safety profiles. Although current evidence indicates no local or systemic adverse effects from PENs [95], however, whether all PENs types can effectively reach target cells and exert biological effects without toxicity requires further investigated. Additionally, PENs concentration and purity from different plant sources during isolation need careful attention [196]. Systemic toxicity assessments of long-term topical PENs use, including chronic toxicity, allergenicity, and dermal accumulation, is critical for dermatological applications.

Stability limitations and storage requirements for topical applications

As delivery vehicles for dermatological treatments, PENs must maintain their structural and functional integrity under practical conditions. Current evidence indicates that PENs are temperature-sensitive, necessitating storage at −80 °C to preserve their bioactivity. Freeze-thaw cycles can disrupt the integrity of their lipid bilayer, thereby compromising drug-loading efficiency [139, 197], and may also lead to the aggregation of exosomes. Given the real-world demands for ambient-temperature transport and prolonged shelf life in topical formulations, cryoprotectants such as trehalose, DMSO, and glycerol have been proposed to enhance stability [63], but little is known about the significance or effectiveness of these methods [198]. Furthermore, the effects of various storage modalities on the transdermal delivery capacity and therapeutic effectiveness of transdermal patches (PENs) remain poorly understood. There is a notable absence of standardized pharmacokinetic-pharmacodynamic (PK-PD) parameters, particularly concerning stability data under ambient conditions. Since topical dermatological formulations typically require storage at ambient conditions and have extended shelf lives, it is crucial that future research focuses on developing strategies to enhance stability and create delivery systems specifically designed for practical application contexts.

Challenges in PEN drug delivery therapy

Research on PENs as drug delivery vehicles remains relatively limited, and their therapeutic potential has yet to be fully elucidated. Despite their favorable biocompatibility, PENs face significant challenges regarding cost efficiency, production yield, drug-loading capacity, tissue targeting precision, and controlled release performance. Lipidomic analyses indicate that PENs are predominantly composed of phospholipids, lacking the cholesterol and sphingomyelin that characterize mammalian exosomes. However, systematic investigations into how these distinct lipid profiles influence cellular uptake efficiency remain scarce [199]. Compared to mammalian exosomes, PENs exhibit inherent tissue tropism, particularly toward the liver and intestines [93, 94, 137]. Nonetheless, there is a deficiency of ligands suitable for efficient cell targeting. To enhance target specificity, the application of RNA nanotechnology is proposed. The post-biogenesis approach within RNA nanotechnology employs gelatin-like exosome nanoparticles to adjust the angle and orientation of the RNA structure, thereby facilitating the presentation of ligands on the exosomal surface [200–202]. This methodology facilitates accurate targeting through the incorporation of RNA nanostructures into exosomes, which are subsequently modified to present specific ligands on their exterior. A research investigation utilized RNA nanotechnology to develop a four-way junction (4WJ) RNA structure, which was co-loaded with paclitaxel (PTX) and miR-122. By functionalizing the surface of the exosomes with N-acetylgalactosamine (GalNAc), the system achieved targeted recognition of the asialoglycoprotein receptor (ASGP-R) present on hepatocellular carcinoma cells [203]. Future research may expand upon these strategies to create PEN-based targeted systems specifically for cutaneous disorders. Additionally, although the oral route of administration provides considerable convenience, it is significantly hindered by reduced drug bioavailability due to extensive first-pass metabolism. In contrast, intravenous administration ensures substantial therapeutic effectiveness but raises safety concerns in non-clinical environments. The transdermal delivery route may offer a viable compromise; however, its application within penetrative drug delivery systems (PENs) has not been thoroughly investigated. Subsequent studies should systematically evaluate various administration routes for the treatment of different dermatological conditions to enhance drug delivery methodologies.

Others

Current research on PENs faces significant challenges in standardization. Variations in botanical sources, extraction protocols, size distribution, and biomarker expression across studies hinder the comparability of results and their translational value. Establishing unified quality control criteria and preclinical evaluation frameworks is imperative for advancing manufacturing standardization.Furthermore, the expanding application of PENs for long-term topical use and exogenous therapeutic interventions raises ethical concerns, including the legal sourcing of plant materials, sustainable harvesting practices, and regulatory compliance for cross-border biotransport. It is essential to incorporate these concerns into both research and clinical frameworks to ensure the ethical and biosafe implementation of interventions. The diverse characteristics of dermatological conditions, along with individual patient factors such as skin barrier integrity and immune status, present challenges for the application of PEN-based therapies. Natural products, which possess mechanisms and bioactive components that are not fully understood, necessitate the development of personalized treatment strategies specifically designed to address individual pathophysiological conditions. Currently, clinical practice lacks comprehensive algorithms that optimize PEN regimens based on multi-omics profiling, including proteomic and lipidomic signatures. Future research should prioritize the establishment of standardized production pipelines in conjunction with precision medicine methodologies. The integration of big data analytics, particularly artificial intelligence-driven patient stratification and machine learning algorithms for predicting drug-carrier compatibility, has the potential to revolutionize the applications of PEN in dermatology. This integration could facilitate the development of patient-specific therapies that exhibit enhanced efficacy and safety profiles.

Conclusion and perspectives

In conclusion, this review systematically examines the biogenesis, isolation and purification techniques, and compositional characteristics of PENs. It offers a comprehensive comparison of the structural and functional differences between exosomes derived from plants and those from animals. Additionally, we analyzed the various delivery methods for PENs as drug carriers, including oral administration, transdermal absorption, and injectable formulations. Mechanistically, PENs exhibit synergistic therapeutic effects in treating dermatological conditions such as skin aging, alopecia, hyperpigmentation, and impaired wound healing. These effects are facilitated by variousmechanisms, including the scavenging of ROS, the modulation of anti-inflammatory signaling pathways, and the regulation of cytokine expression. Taken together, these diverse mechanisms underscore the considerable potential of PENs in promoting and maintaining skin health.

Despite their significant therapeutic potential, the current research of PENs primarily relizes on in vitro and in vivo animal models, revealing a notable deficiency in clinical studies and insufficient data. Therefore, it is essential to address the technical and regulatory challenges that impede their clinical translation. To date, only four clinical trials focusing on PENs have been registered on Clinicaltrials.gov (NCT01668849, NCT04879810, NCT01294072, NCT03493984; IRCT20200127046282N53) (Table 5), and these trials have achieved their primary endpoints. However, substantial data from these trials remain unavailable, likely due to ongoing studies, pending submissions of results, or the lack of mandatory data-sharing policies. Considering the limited number of clinical trials involving PENs, addressing current challenges and promoting further clinical investigations to substantiate their therapeutic potential are essential [204].

Table 5.

Summary of clinical trials involving PENs for various medical conditions

Applications	Study details	Last update posted	Status	Interventions	Study type	Phase	Study results
Head and Neck Cancer/Oral Mucositis	(NCT01668849) Study on the ability of grape exosomes to alleviate oral mucositis induced by chemoradiation in head and neck cancer patients	2022/8/9	Completed	Dietary_Supplement: Grape Extract/Drug: Lortab, Fentanyl Patch, Mouthwash	Interventional	PHASE 1	NO
Irritable Bowel Disease	(NCT04879810) study on the ability of ginger exosomes ± curcumin to alleviate symptoms of inflammatory bowel disease (IBD)	2022/11/3	Completed	Procedure: Sigmoidoscopy And Biopsy, Blood Work	Interventional	Not Applicable	NO
Colon Cancer	(NCT01294072) study on the ability of plant exosomes to deliver curcumin to normal and malignant colon tissue	2023/12/20	Recruiting	Dietary_Supplement: Curcumin/Dietary_Supplement: Curcumin Conjugated With Plant Exosomes/Other: No Intervention	Interventional	Not Applicable	NO
Polycystic Ovary Syndrome	(NCT03493984) study on the ability of ginger or aloe exosomes to alleviate insulin resistance and chronic inflammation in patients with polycystic ovary syndrome (PCOS)	2021/3/16	Withdrawn	Other: Ginger Exosomes/Other: Aloe Exosomes/Other: Placebo	Interventional	Not Applicable	NO

The utilization of PENs in treating dermatological conditions faces several significant obstacles, such as the lack of standardized methods for isolation and purification, the prohibitive costs associated with large-scale production, the insufficient understanding of in vivo metabolic processes, and the absence of well-established frameworks for assessing clinical efficacy and safety. We emphasize the necessity of interdisciplinary collaboration among clinicians, cell biologists, pharmaceutical scientists, clinical trial specialists, and bioinformaticians. Such synergy will advance the understanding of PEN biological mechanisms, optimize extraction and purification techniques, and foster innovative therapeutic strategies. Future investigations should focus on refining the preparation protocols for PENs, elucidating their mechanistic actions, and developing comprehensive clinical validation systems. These efforts are essential for promoting the effective transition of PENs from preclinical studies to clinical applications in dermatological therapy.

Author contributions

H.L. was responsible for drafting the original manuscript. T.D. contributed to all the illustrations. C.D., F.Y. assisted in collecting references. Q.Z. offered financial support and assisted in collecting references. C.G. provided financial assistance and contributed to the outlining and revision of the manuscript. W.W. provided funding support, as well as outlining and supervising the manuscript. All authors reviewed the manuscript.

Funding

This research was funded by the Zhejiang Provincial Natural Science Foundation of China (grant No. LKLY25H180015), the Science and Technology Major Project of Zhejiang Province and the State Administration of Traditional Chinese Medicine (grant No. GZY-ZJ-KJ-23035), the Hangzhou Public Welfare Scientific Research Guidance Program the Field of Agriculture and Social Development (grant No. 20241029Y034) and the Hangzhou Medical Key Discipline Construction Project (grant No. [2025]36 − 7).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.