Current Research on Aloe-Derived Extracellular Vesicles in Injury Repair

Introduction

Extracellular vesicles (EVs) are nanoscale, lipid bilayer-enclosed particles secreted by cells that carry bioactive cargo such as proteins, lipids, and nucleic acids. They play essential roles in mediating intercellular communication across diverse biological systems. In recent years, plant-derived extracellular vesicle-like nanoparticles (PDVLNs) have emerged as a promising alternative to their mammalian counterparts. PDVLNs offer distinct advantages, including abundant sources, lower immunogenicity, scalable production from agricultural resources, and inherent biocompatibility, positioning them as attractive candidates for therapeutic development.^1,2

Tissue injury repair, encompassing processes like wound healing and resolution of internal organ damage, remains a central challenge in clinical medicine. Efficient repair requires a precisely coordinated cascade of events, including inflammation control, oxidative stress mitigation, cell proliferation, migration, and extracellular matrix remodeling. While numerous therapeutic agents have been explored, many face limitations such as poor stability, low bioavailability, and off-target effects, highlighting the need for novel, targeted delivery platforms.

The genus Aloe (family Liliaceae), with a history of medicinal use spanning millennia, is a rich source of bioactive compounds. Modern research confirms that its constituents, including polysaccharides, anthraquinones (eg, aloin, aloe-emodin), flavonoids, and phenolic acids, contribute to potent antioxidant, anti-inflammatory, and immunomodulatory activities relevant to tissue repair.^3–5 However, the therapeutic efficacy of crude aloe extracts can be limited by rapid degradation, inconsistent absorption, and the complex, sometimes antagonistic, mixture of compounds. This is where the unique properties of aloe-derived extracellular vesicles (A-EVs) become significant. These natural nanoparticles serve as sophisticated delivery systems, encapsulating and protecting aloe’s bioactive cargo. Thus, A-EVs represent more than a refined extract; they constitute a natural, membrane-based delivery system that enhances the stability and bioavailability of key therapeutic molecules and may facilitate targeted delivery, thereby amplifying the reparative potential of aloe beyond that of traditional extracts.^6,7

Within the broader landscape of PDVLNs, A-EVs exhibit a distinctive profile. Compared to vesicles from other well-studied plants like ginger or grapefruit, A-EVs are uniquely enriched in specific anthraquinones and flavonoids, which may confer a synergistic combination of antioxidant and anti-inflammatory actions highly suited for the multiphase demands of injury repair.^8,9 Moreover, A-EVs have been studied in multiple preclinical models relevant to tissue repair, such as skin wounds and colitis, providing a valuable translational foundation.¹⁰

Despite rapid progress, the field faces significant challenges that must be addressed to translate promise into practice. Key issues include a lack of standardized isolation protocols leading to variable EV preparations, incomplete understanding of their in vivo biodistribution and pharmacokinetics, and only partially elucidated molecular mechanisms of action. Furthermore, while their potential as natural nanocarriers for drug delivery is recognized, research into engineering A-EVs for enhanced targeting or combination therapies remains in its infancy.

Therefore, this review aims to: (1) systematically summarize current methods for the isolation and characterization of A-EVs, critically evaluating their strengths and limitations; (2) synthesize the evidence for their multifaceted roles in injury repair—antioxidant, pro-proliferative, anti-inflammatory, and extracellular matrix-modulating—with a focus on underlying molecular pathways such as the Nrf2/HO-1 and NF-κB axes; (3) place A-EVs in the context of other PDVLNs, discussing their comparative advantages and translational potential; and (4) identify critical knowledge gaps, technical hurdles, and future directions, including standardization efforts, mechanistic depth, and their emerging role in nanomedicine as engineered therapeutic carriers. By providing this comprehensive and critical analysis, we seek to clarify the current state of the science and chart a course for the rational development of A-EV-based therapies for tissue repair.

Isolation and Characterization of Aloe-Derived Extracellular Vesicles

Isolation Methods

Obtaining high-purity, high-quality extracellular vesicles (EVs) is fundamental for downstream experimentation. At present, differential centrifugation remains the most commonly used method for isolating aloe-derived EVs.^11,12 This technique separates cellular debris, organelles, and large particles based on their sedimentation rates under gradually increasing centrifugal forces, ultimately yielding nanoscale vesicles. Differential centrifugation is simple, reproducible, and suitable for processing large sample volumes, making it widely used in early studies of plant EVs. However, it has notable limitations: ultracentrifugation is time-consuming and sensitive to operational variability; repeated centrifugation can lead to sample loss and reduced EV activity; and co-precipitation of non-vesicular proteins and other impurities may interfere with functional analyses.^13,14 To address these issues, researchers have explored complementary techniques to improve purity and yield, though each approach entails trade-offs between scalability, cost, and biological fidelity.

Differential Centrifugation Combined with Ultrafiltration

Ultrafiltration (UF) is a pressure-driven separation technique based on the size-selective properties of semipermeable membranes. A patented protocol for isolating aloe-derived nanovesicles integrates differential centrifugation with ultrafiltration: sequential centrifugation at 350–500×g, 500–1500×g, 1500–2500×g, and 12,000–15,000×g first removes large impurities from aloe extracts. The resulting supernatant containing EVs is then concentrated using ultrafiltration tubes (1500–2500×g) to remove small molecules and excess water. Finally, EVs are pelleted by ultracentrifugation at 100,000–150,000×g and resuspended in 1×PBS containing protease inhibitors (1.5 μg/mL) and 25 mM trehalose. Incorporating ultrafiltration improves both purity and concentration while better preserving EV bioactivity.¹⁵ However, the dependence on single-use UF cartridges increases cost and limits scalability.¹⁶ Importantly, this hybrid approach reduces contamination from polysaccharides—a major challenge in plant EV isolation due to the high mucilage content in aloe gel—thereby enhancing compatibility with downstream omics analyses.

Polymer-Based Precipitation

Polymer precipitation relies on the ability of polymers to create a hydrophobic microenvironment around EVs, reducing their solubility. Kim et al employed polyethylene glycol (PEG) precipitation after differential centrifugation: the post-centrifugation supernatant was mixed with an equal volume of 2× PEG and incubated at 4 °C, followed by centrifugation at 1500×g. The pellet was washed with PBS and resuspended to obtain aloe-derived EVs. Among different conditions, incubation with 8% PEG for 16 hours produced the highest purity AS-EVs.⁶ This method is cost-effective and suitable for large sample volumes, but residual polymers may remain in the final preparation, potentially interfering with cell-based assays or mass spectrometry.¹⁷ Notably, PEG-based isolation has been shown to co-precipitate exosome-like structures along with non-vesicular nanoparticles in plant systems, raising concerns about specificity when interpreting functional data.

Tangential Flow Filtration (TFF)-Assisted Centrifugation

Tangential flow filtration (TFF) uses pressure to drive sample flow tangentially across a membrane surface, allowing selective passage of smaller components while minimizing membrane fouling.¹⁸ In one study extracting EVs from aloe peel, differential centrifugation was combined with TFF: after initial centrifugation, samples were passed through a 0.22 µm filter and concentrated using a 300-kDa TFF membrane.¹⁹ TFF enables efficient processing of large volumes and reduces clogging, but it requires specialized instrumentation, increasing both cost and operational complexity.¹⁶ Recent comparative studies suggest that TFF yields EV preparations with lower protein-to-particle ratios than ultracentrifugation alone, indicating higher purity—particularly valuable for in vivo applications where immune activation by contaminants must be minimized.²⁰

Critical Perspective on Methodological Variability

The lack of standardized protocols for plant EV isolation remains a major barrier to cross-study comparability. Unlike mammalian EVs, plant EVs are embedded in complex matrices rich in phenolics, polysaccharides, and secondary metabolites, which complicate purification—a challenge not unique to aloe but common across the field of plant-derived extracellular vesicles (PELNs).²¹ Moreover, no consensus exists on optimal tissue sources (gel vs. peel), harvest timing, or storage conditions—all factors influencing EV yield and composition.²² This heterogeneity contributes to the wide size distribution reported across studies (see Table 1) and underscores the urgent need for community-wide validation of reference materials and benchmarking procedures aligned with MISEV2018/2023 guidelines.²³

Table 1 Summary of Reported EV Morphology and Size from Various Aloe Species

Characterization Techniques

According to the guidelines of the International Society for Extracellular Vesicles (ISEV), aloe-derived EVs are typically characterized in terms of morphology, particle size distribution, and molecular composition.²⁴ Consistent with these recommendations, current studies employ multimodal approaches.

Morphology and Particle Size

Transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM) are essential for morphological characterization. Across multiple studies, aloe EVs isolated from different species, tissues, and centrifugal conditions consistently display classical EV morphology: irregular spherical or discoid vesicles with smooth surfaces and a phospholipid bilayer. Particle size and concentration are commonly determined using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Published data indicate that aloe-derived EVs generally range from 100 to 300 nm in diameter,^{15,20,25–28} providing a structural basis for understanding their biological behavior. After summarizing the general morphological features and size distribution of aloe-derived EVs, the specific findings reported across different studies are compiled in Table 1.

Composition Analysis and Safety Considerations

Protein composition is typically analyzed using BCA assays, SDS–PAGE, and mass spectrometry. Aloe-derived nanovesicles contain common plant vesicle-related proteins, such as histone H4, heat shock protein 70, and actin.^29,30 Notably, four proteins enriched in aloe EVs—glutathione S-transferase P, serum transferrin, RNA polymerase σ factor sigE, and UTP–glucose-1-phosphate uridylyltransferase—were not detected in crude aloe extracts, suggesting active sorting mechanisms.²⁰ Lipidomic analyses have identified multiple phospholipid species, including phosphatidylcholine and phosphatidylethanolamine, consistent with a lipid bilayer structure.²⁰ Small-molecule profiling has revealed anthraquinone compounds such as aloin and aloe-emodin, which possess antioxidant and anti-inflammatory properties. Owing to selective encapsulation, however, their concentrations in EVs are lower than in crude plant extracts.²⁰ This selective packaging implies a regulated biogenesis process, possibly involving ESCRT-independent pathways common in plant cells.²³

Despite widespread use of aloe in traditional medicine, potential toxicity of aloe-derived EVs requires attention. Aloin, a major anthraquinone, is known to cause genotoxicity at high doses in rodent models.³¹ However, current evidence suggests that EV-encapsulated aloin levels are significantly reduced compared to whole extracts, potentially mitigating risk.³² No adverse effects have been reported in murine wound or colitis models treated with aloe EVs at therapeutic doses (≤50 μg/mL or ≤1×10⁹ particles/mL).^{6,12,15,17–19} Nevertheless, comprehensive toxicokinetic studies—including long-term biodistribution, immunogenicity, and organ accumulation—are still lacking and represent a key knowledge gap before clinical translation.

Biological Functions and Mechanistic Insights of Aloe-Derived Extracellular Vesicles

Antioxidant Activity

Oxidative stress is a key pathological mechanism in tissue injury. Excessive reactive oxygen species (ROS) induce lipid peroxidation, protein carbonylation, and DNA damage, ultimately impairing cellular function.³³ Aloe-derived EVs exhibit potent antioxidant capacity through two complementary mechanisms. First, phenolic compounds enriched in aloe EVs—including aloe-emodin and aloin—can directly scavenge ROS.^5,34 Second, aloe EVs modulate intracellular oxidative stress pathways to enhance cellular defense.

Using ABTS radical scavenging assays and intracellular ROS inhibition assays, in vitro studies demonstrated that cortex-derived aloe EVs (rAEVs) clear ABTS radicals in a dose-dependent manner, reaching a scavenging efficiency of 93.17% at 30×10⁵ mL⁻¹. rAEVs also suppressed intracellular ROS formation, decreasing ROS fluorescence by 52.55% at a protein concentration of 50 μg/mL.²¹ The transcription factor Nrf2 is central to the antioxidant defense system, triggering the expression of cytoprotective enzymes such as HO-1, SOD, and CAT upon activation.^35,36 Aloe peel–derived EVs (A-EVs) markedly activated the Nrf2 signaling pathway in human keratinocytes (HaCaT) and dermal fibroblasts (HDF), significantly reducing cellular ROS levels by over 40% and increasing cell viability by 25%.¹⁹ Consistently, EVs derived from several Aloe species (VNVs, ANVs, SNVs) lowered the expression of the oxidative damage marker 3-NT in the colon and small intestine of mice with acute colitis, confirming their antioxidant role in vivo.²⁴ The multifunctional roles of A-EVs in orchestrating tissue repair, from cargo delivery to coordinated cellular responses, are summarized in Figure 1.

Figure 1 Schematic overview of the isolation, composition, and multifaceted mechanisms of aloe-derived extracellular vesicles (A-EVs) in tissue injury repair. (A) Isolation workflow: Fresh aloe leaves undergo homogenization, followed by sequential differential centrifugation to remove cellular debris and large particles. The crude EV-containing supernatant is further purified using techniques such as ultrafiltration (UF), polyethylene glycol (PEG) precipitation, or tangential flow filtration (TFF) to obtain purified A-EVs. (B) Key cargo components: A-EVs are enriched with bioactive molecules including anthraquinones (eg, aloin, aloe-emodin), flavonoids, specific proteins (eg, heat shock proteins, actin), lipids, and potential nucleic acids. (C) Multimodal reparative actions: Upon delivery to injury sites, A-EVs orchestrate repair through interconnected mechanisms: 1) Antioxidant: Activating the Nrf2/HO-1 pathway to scavenge ROS. 2) Anti-inflammatory and Immunomodulation: Promoting macrophage polarization from M1 to M2 phenotype and inhibiting the NF-κB pathway. 3) Proliferation & Migration: Enhancing fibroblast and keratinocyte motility, potentially via cytoskeletal remodeling. 4) ECM Regulation: Inhibiting TGF-β1-induced myofibroblast differentiation and restoring tight junction proteins. 5) Pyroptosis Induction: Triggering Gasdermin-mediated pyroptosis in dysfunctional cells (eg, in tumor microenvironments), releasing DAMPs and cytokines (eg, IL-18) to stimulate adaptive immunity.

Promotion of Cell Proliferation and Migration

Cell proliferation and migration are critical events governing wound repair, directly determining the rate and quality of tissue regeneration.³⁷ Multiple studies have shown that aloe-derived EVs significantly enhance these processes. Using WST-8 assays and scratch-wound experiments, AS-EVs from Aloe saponaria were shown to increase human dermal fibroblast proliferation by 2.3-fold and migration by more than threefold at 5×10⁹ particles/mL, exhibiting strong dose dependence. These results suggest that higher concentrations of AS-EVs may be particularly beneficial during chronic wound repair.¹²

Similarly, rAEVs promoted the proliferation and migration of human epidermal keratinocytes (HaCaT) and human umbilical vein endothelial cells (HUVECs), with both effects increasing in a concentration-dependent manner. At 50 μg/mL, rAEVs increased HUVEC migration to 3.9 times that of controls.⁶ Scratch assays by Kim et al further showed that A-EVs (1×10⁹ particles/mL) achieved 70% scratch closure in HaCaT cells and 100% in HDFs after 24 hours.¹⁵

Although the precise mechanisms remain incompletely defined, cytoskeletal remodeling—particularly actin dynamics—is essential for cell motility. Thus, aloe EVs may enhance cell migration not only by improving the microenvironment through anti-inflammatory and antioxidant actions, but also by modulating actin cytoskeletal reorganization.^3,38

Anti-Inflammatory Activity

Inflammation is a protective response essential for early wound defense, but persistent or excessive inflammation delays healing and can lead to chronic inflammatory disorders.^39,40 Aloe-derived EVs exhibit notable anti-inflammatory activity via immunomodulation and inhibition of pro-inflammatory signaling pathways.

In an acute colitis mouse model, aloe nanovesicles significantly reduced the expression of inflammatory markers (p-IκB, p-NF-κB) and oxidative stress markers (COX-2, 3-NT) in colonic and intestinal tissues.¹⁸ Ramírez et al further evaluated peel-derived vesicles (AVpNVs) in LPS-stimulated RAW264.7 macrophages, THP-1 macrophages, and HaCaT cells. High-dose AVpNVs (500 particles/cell) markedly reduced IL-1β and TNF-α secretion, outperforming both low-dose EVs and the aloe flavonoids quercetin and kaempferol.¹⁸

Zhou et al demonstrated that aloe EVs are efficiently internalized by macrophages, promoting a shift from pro-inflammatory M1 (iNOS⁺) to anti-inflammatory M2 (Arg-1⁺) polarization, accompanied by decreased pro-inflammatory cytokines and increased IL-10 secretion.¹⁷

There is emerging evidence that aloe EVs may exert anti-inflammatory effects partly through crosstalk between the Nrf2 and NF-κB pathways. A-EVs activate Nrf2/HO-1 signaling under oxidative stress,¹⁵ and Nrf2 activation is known to suppress NF-κB through reduced ROS generation and competitive inhibition of transcriptional activity.^41,42 Thus, aloe EVs may attenuate inflammation by indirectly limiting NF-κB nuclear translocation.

Regulation of Extracellular Matrix (ECM) Metabolism

The extracellular matrix (ECM) is essential for maintaining tissue structure and function, and its balanced remodeling is crucial for successful wound repair.^43,44 Fibroblasts, which synthesize ECM components and differentiate into myofibroblasts during healing, contribute to collagen deposition and wound contraction. However, persistent myofibroblast activation can lead to excessive ECM accumulation and pathological scarring.⁴⁵

Beyond promoting fibroblast proliferation and migration, aloe EVs help modulate ECM remodeling by suppressing myofibroblast differentiation. AVpNVs significantly inhibited TGF-β1–induced myofibroblast transformation in neonatal human dermal fibroblasts (HDNFs), reducing α-SMA expression.¹⁸ In gastrointestinal inflammation models, aloe nanovesicles also preserved ECM integrity by restoring tight junction proteins such as claudin-4 and ZO-1, thereby strengthening mucosal barrier function.¹⁷

Induction of Programmed Cell Death and Immune Activation

Emerging evidence positions aloe-derived EVs as regulators of programmed cell death, a critical process in removing damaged cells during injury and cancer. A landmark study demonstrated that aloe vera-derived EV-like particles, particularly those isolated by ultracentrifugation, exhibit potent anti-tumor activity in pancreatic cancer models by inducing pyroptosis, a lytic and inflammatory form of programmed cell death.⁴⁶ Mechanistically, aloe vera-derived EV-like particles triggered a significant increase in intracellular ROS, which in turn activated caspases-1/3/7/9 and cleaved their downstream effector proteins, Gasdermin D and E (GSDMD/E), leading to characteristic pyroptotic cell swelling and membrane rupture.³¹

This pyroptosis induction has profound implications for injury repair and immune modulation. The lytic cell death releases damage-associated molecular patterns (DAMPs) and inflammatory cytokines such as IL-18, which can stimulate an adaptive immune response.⁴⁷ This finding creates a novel mechanistic link between the established antioxidant (ROS-modulating) capacity of aloe EVs and their immunomodulatory functions. It suggests that in contexts like chronic wounds or tumor microenvironments, aloe EVs may not only suppress detrimental inflammation via the Nrf2/NF-κB axis but also actively remodel the tissue by eliminating dysfunctional cells and converting the immune milieu from a suppressed (“cold”) state to a more active (“hot”) one, thereby facilitating clearance and regeneration.⁴⁸ The reparative effects of A-EVs, as demonstrated across a range of in vitro and in vivo injury models, are comprehensively summarized in Table 2.

Table 2 Summary of Biological Activities of Aloe-Derived Extracellular Vesicles in Various Injury Models

Comparative Advantages, Biodistribution, and Emerging Applications

Why Aloe? Unique Attributes Among Plant EV Sources

Compared to EVs from other widely studied medicinal plants (eg, ginger, grapefruit, broccoli), aloe-derived EVs offer a distinctive combination of properties relevant to healing.⁴⁹ They inherit the longstanding safety profile and traditional use of Aloe vera in wound care. Their cargo is uniquely enriched with both antioxidant anthraquinones (aloin, aloe-emodin) and anti-inflammatory lipids/proteins, supporting a multi-targeted approach to repair.^25,50 Furthermore, their physical stability, potentially due to membrane composition, and possible mucoadhesive properties make them suitable for topical and oral delivery.^10,15 The distinctive profile of A-EVs becomes evident when compared to other well-studied plant EVs, as detailed in Table 3.

Table 3 Comparative Analysis of Aloe-Derived Extracellular Vesicles with Other Representative Plant-Derived EVs

Pharmacokinetics, Biodistribution, and Therapeutic Carrier Potential

The in vivo journey of aloe EVs remains largely uncharted, a common gap in the PELNs field.⁵¹ While evidence confirms oral aloe EVs reach the gut and exert effects,^17–19 their systemic distribution, cellular targeting specificity, and clearance pathways after topical or intravenous administration are unknown. Addressing this via tracking studies is crucial for clinical translation.

Concurrently, the innate biocompatibility, low immunogenicity, and nano-scale structure of aloe EVs make them compelling natural drug delivery vehicles.^11,13,52 The broader PELNs field has demonstrated remarkable success in loading chemotherapeutic agents (eg, paclitaxel), nucleic acids (siRNA, miRNA), and anti-inflammatory drugs into vesicles from ginger or grapefruit, enhancing drug stability, targeting, and efficacy while reducing off-target toxicity.⁵³ Although similar engineering of aloe EVs is in its infancy, their demonstrated stability and bioactive membrane render them an excellent candidate platform for such applications. Future directions include surface functionalization for active targeting and embedding within hydrogels for sustained release in chronic wounds.⁴⁷

Conclusion and Perspectives

Current evidence demonstrates that aloe-derived extracellular vesicles (A-EVs) exert therapeutic effects across multiple injury models. They accelerate full-thickness wound closure in mice (from 42.42% in controls to 75.29%), mitigate UV-induced fibroblast damage during photoaging, and reduce body weight loss and intestinal injury in DSS-induced acute colitis models.^10,12,19 Notably, A-EVs also promote regeneration of damaged hair follicles, suggesting a capacity not only for structural repair but also for functional restoration. Beyond their intrinsic bioactivity, A-EVs exhibit distinct advantages over other plant-derived EVs: their natural enrichment in redox-modulating anthraquinones (eg, aloin, aloe-emodin), cytoskeleton-regulating proteins, and mucoadhesive surface properties endow them with a unique ability to simultaneously resolve oxidative stress, modulate immune responses, and enhance epithelial cell migration—key requirements in complex, non-healing wounds such as diabetic ulcers or radiation-induced tissue damage.^41,50 This multifunctionality positions A-EVs not merely as alternatives to synthetic nanocarriers, but as irreplaceable biological platforms for regenerative applications where coordinated signaling is essential. Nevertheless, the path from bench to bedside remains obstructed by critical translational gaps. First, the lack of consensus on isolation methods (eg, differential ultracentrifugation versus size-exclusion chromatography) results in heterogeneous preparations with variable cargo and bioactivity, undermining reproducibility.^16,40 Second, scalable, GMP-compliant production of A-EVs from agricultural sources has not yet been demonstrated; batch consistency, endotoxin control, and long-term stability under storage conditions remain unresolved.⁴⁵ Third, while preclinical data are compelling, pharmacokinetic profiles, tissue biodistribution, and chronic toxicity in higher mammals are largely unknown—essential prerequisites for regulatory approval. To realize the clinical potential of A-EVs, we propose a focused research roadmap: (i) establish standardized protocols for A-EV isolation, characterization, and potency assays aligned with MISEV guidelines; (ii) deconvolute the contribution of individual cargo components (eg, via proteomic/lipidomic profiling coupled with functional knockdown studies) to identify minimal effective signatures; (iii) engineer A-EVs for enhanced targeting (eg, surface modification for homing to inflamed endothelium) or combination therapy (eg, co-loading with growth factors or siRNA); and (iv) prioritize validation in clinically relevant models of chronic wounds, inflammatory bowel disease, and mucosal injury—conditions with high unmet medical need and limited therapeutic options. In summary, A-EVs represent a convergence of traditional herbal wisdom and modern nanomedicine. With strategic investment in standardization, mechanistic depth, and translational validation, they hold realistic promise as next-generation biologics for regenerative medicine—not as panaceas, but as precisely defined, naturally derived therapeutics tailored to the dynamic demands of tissue repair.