Aim: This study investigates the change in profiles of miRNAs in extracellular vesicles released during Toxoplasma gondii (T. gondii) infection. T. gondii has been implicated in host behavioural modifications and neuroinflammatory responses, yet the molecular mechanisms involved in these changes remain poorly understood. Extracellular vesicles, involved in intercellular communication, play an important role in host-pathogen interactions, particularly through the transfer of microRNAs (miRNAs); however, the impact of extracellular vesicle miRNAs in T. gondii infection remains largely unexplored.

Methods: Human BE(2)-M17 neuronal cells were infected with Toxoplasma gondii to investigate infection-induced changes in extracellular vesicle (EV) miRNA content. EVs from infected and control cultures were isolated, characterised, and subjected to miRNA extraction followed by next-generation sequencing and differential expression analysis using standard bioinformatic pipelines. Predicted miRNA targets were integrated across multiple databases and analysed for enriched pathways to identify neuronal regulatory networks.

Results: Pathway network analysis identified key neurobiological pathways, including neuroplasticity, neurotransmission, and neuroinflammation in high-confidence miRNA targets with gene enrichment of neurotrophin and long-term depression and long-term potentiation, which may underlie parasite-induced alterations in neural function. Bioinformatic analysis of extracellular vesicle miRNA profiles from infected and uninfected neuronal cells revealed a set of miRNAs including hsa-miR-4645-3p with significant upregulation in response to infection.

Conclusion: These findings suggest that T. gondii modulates host neuronal processes through extracellular vesicle-mediated miRNA transfer, providing a potential mechanistic link between infection and parasite-associated cognitive and neuropsychiatric disturbances.

A recent study on Cell Reports Medicine by Wang et al. introduces a hybrid exosome platform - selenized neural stem cell-derived exosomes (SeNExo) - that couples the biological functionality of neural stem cell exosomes with the antioxidant power of ultrasmall nanoselenium. SeNExo crosses the blood-brain barrier via apolipoprotein E (APOE)-lipoprotein receptor-associated protein-1 (LRP1) interaction, scavenges reactive oxygen species, and restores glial-neuron homeostasis. It demonstrates potent therapeutic efficacy in both traumatic brain injury and spinal cord injury mouse models. This work highlights a promising direction for engineering multifunctional, cell-free nanotherapeutics for central nervous system repair.

Mesenchymal stromal/stem cell-derived extracellular vesicles (MSC-EVs) have emerged as promising acellular therapeutics in regenerative medicine, offering a safer and more controllable alternative to whole-cell therapies. Their therapeutic efficacy, however, is highly dependent on their molecular cargo, which reflects the physiological state and environmental conditions of the parent MSCs. Priming of mesenchymal stromal/stem cells (MSCs) with defined stimuli such as hypoxia, inflammatory cytokines, 3D culture systems, biomaterials, or pharmacological agents has been increasingly employed to enhance extracellular vesicle (EV) bioactivity. These strategies modulate EV content, enriching vesicles with regenerative, immunomodulatory, angiogenic, and antioxidant factors. For instance, hypoxic priming activates hypoxia-inducible factor-1α-driven gene expression, promoting the packaging of angiogenic and anti-inflammatory molecules, while cytokine-based priming upregulates immunosuppressive proteins and regulatory microRNAs. Similarly, 3D culture mimics aspects of the native tissue microenvironment, augmenting the secretion of EVs with enhanced reparative potential. Emerging combination-based approaches synergize these effects, generating EVs with superior therapeutic profiles. Despite encouraging preclinical data, translation to clinical application is challenged by variability in MSC sources, priming conditions, and EV isolation methods. Standardization of protocols, validated potency assays, and regulatory harmonization are critical for clinical advancement. This mini-review summarizes current priming strategies, the underlying mechanisms influencing EV cargo, and their functional implications in disease models, while highlighting key barriers and future directions for the clinical translation of primed MSC-EV therapies.

Aim: To investigate the therapeutic potential and underlying mechanism of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in treating hepatorenal syndrome (HRS), a condition lacking therapies for multi-organ damage.

Methods: EVs were isolated from human umbilical cord MSCs and characterized by transmission electron microscopy, nanoparticle tracking analysis, and proteomics. A murine model of HRS, induced by bile duct ligation (BDL), was established, and mice received intravenous MSC-EVs treatment. Therapeutic efficacy was assessed through histopathology, serum biochemistry, and analysis of necroptosis, inflammation, and fibrosis markers.

Results: Proteomic profiling of MSC-EVs revealed significant enrichment of proteins involved in renal processes, anti-fibrosis, and immune regulation. In BDL-induced HRS mice, MSC-EVs treatment demonstrated potent multi-organ protective effects. This was evidenced by alleviation of hepatic necroptosis and renal tubular injury, downregulation of interleukin-17 expression, and concurrent attenuation of fibrosis in both liver and kidney tissues. Consequently, significant improvements in hepatic and renal function markers were observed.

Conclusion: MSC-EVs represent a novel and effective cell-free nanotherapeutic strategy for HRS. They confer protection through multi-faceted mechanisms, including inhibition of necroptosis, immune reprogramming, and fibrosis resolution, offering a promising paradigm for the treatment of multi-organ failure.

The existence of small vesicles released by cells into the extracellular space was first documented over 40 years ago. These nanoparticles, now recognized as extracellular vesicles (EVs), were originally defined as “cellular dust” reflecting the early belief that their primary function was to dispose of cellular waste. Nowadays, it is widely acknowledged that EVs make a fundamental contribution to intercellular communication, being capable of transporting biologically active molecules, including proteins and nucleic acids, which regulate both physiological and pathological processes. Their involvement in various diseases, particularly cancer, has been well documented. EVs influence tumor development, progression, and therapeutic response, and have therefore been considered potential diagnostic and prognostic biomarkers. In this review, we focus on the contribution of EVs in modulating tumor cell metabolism and the tumor microenvironment. Specifically, we describe how EVs promote angiogenesis, induce the transformation of fibroblasts into cancer-associated fibroblasts, and influence extracellular matrix remodeling. Additionally, we explore their contribution to the reprogramming of tumor metabolism, including glycolytic, lipid, and amino acid pathways. We provide an in-depth overview of the key molecules carried by EVs that contribute to these pro-tumorigenic effects and of the underlying mechanisms involved.

Aim: This proof-of-concept study aimed to evaluate the impact of fluorescence-based sorting on the microRNA (miRNA) molecular profile of extracellular vesicles (EVs) derived from adipose-derived mesenchymal stromal cells (ASCs) (ASC-EVs), with a focus on osteoarthritis (OA) as a model disease.

Methods: ASCs from five human donors were characterized by flow cytometry and cultured to collect conditioned media. EV isolation was performed by fluorescence-based sorting using a high-sensitivity cell sorter calibrated for particles ≥ 100 nm. In both ASC-EVs and sorted ASC-EVs (sASC-EVs), EV and MSC markers were analysed by flow cytometry, while size and morphology were assessed via nanoparticle tracking analysis and electron microscopy. Small RNA sequencing was used to profile miRNAs, followed by differential expression and functional enrichment analyses.

Results: EVs displayed comparable size distributions and surface marker profiles before and after sorting. In contrast, small RNA sequencing revealed a marked reduction in the number of detectable miRNAs in sASC-EVs relative to EVs isolated by standard ultracentrifugation (285 vs. 749). Among these, 271 miRNAs were shared between groups, exhibiting a strong correlation in relative abundance and functional enrichment in angiogenesis, inflammation modulation and gene silencing pathways. Nevertheless, differential expression analysis identified 32 upregulated and 6 downregulated miRNAs in sASC-EVs, with 14 miRNAs detected exclusively after sorting. Despite these transcriptional differences, the overall balance between protective and detrimental OA-related miRNAs remained positive and was preserved across both ASC-EVs and sASC-EVs.

Conclusion: While sorting reduces EV-associated miRNA diversity, it retains core functional signals. The difference in recovered miRNAs may be due to lower EV recovery, exclusion of miRNA-bound non-vesicular particles or loss of small EVs undetectable by fluorescence-based sorting techniques. Overall, these preliminary findings highlight a trade-off between purity and complexity, underscoring the importance of optimizing EV isolation protocols for clinical applications.

Ovarian cancer is one of the leading causes of gynecologic cancer-related mortality in women. However, a significant proportion of ovarian cancer cases are only detected at an advanced stage (III or IV) and are complicated to treat because of metastasis to the peritoneum. This challenge is compounded by vague symptoms and insufficient screening methods for early ovarian cancer detection. A promising solution is liquid biopsy, where the presence of biomarkers (proteins, lipids, and nucleic acids) associated with cancer is identified in the blood circulation. This approach facilitates the real-time monitoring of cancer progression and treatment effects in a non-invasive manner. This contrasts with traditional tumor biopsy, where only a small portion of the tumor is sampled, serial sampling of the tumor is impractical, or sometimes, tumor biopsy is not feasible. This review discusses the cell-free and extracellular vesicle components in blood, highlighting their DNA as a target in liquid biopsies for cancer diagnostics, with a specific emphasis on ovarian cancer. It also underscores the need for further research into the biological underpinnings and functional roles of these DNA fragments to integrate them into multi-omics approaches for detailed insights into tumor biology and treatment resistance in ovarian cancer.

Aim: The rising prevalence of opioid use during pregnancy poses serious public health concerns. The placenta is a critical organ during gestation, and opioid exposure can disrupt its function and fetal development. However, the molecular mechanisms through which opioids such as oxycodone affect feto-placental development remain poorly understood. This study aimed to investigate the effects of chronic in-utero oxycodone exposure on the composition and signaling functions of placenta-derived small extracellular vesicles (PSEVs) using a rat model.

Methods: Extracellular vesicles (EVs) were isolated from placental tissue and characterized through nanoparticle tracking analysis, transmission electron microscopy, western blotting, and label-free quantitative proteomics. Bioinformatic enrichment analyses were conducted to evaluate changes in EVs biophysical properties and protein cargo.

Results: Chronic oxycodone exposure significantly altered PSEV characteristics, including particle size distribution and proteomic composition. Among the 456 identified EV proteins, 107 proteins were significantly dysregulated. We found key downregulatory proteins including Atp2a2, Lmna, Tgfb3, Agt, and Sgce, which are crucial for myocardial calcium cycling, nuclear integrity, extracellular matrix remodeling, and blood pressure regulation. These findings indicate disruptions in fetal cardiac programming, particularly hypertrophic and dilated cardiomyopathy pathways. Additionally, enrichment analyses revealed notable perturbations in metabolic processes (e.g., citrate cycle, fatty acid degradation, N-glycan biosynthesis), along with upregulation of vesicle transport and neurodevelopment-related proteins, indicating broader systemic effects on fetal development. While these proteomic findings are robust, further independent validation (e.g., via targeted assays or Western blotting) will be necessary to confirm individual protein-level changes.

Conclusion: These results highlight PSEVs as sensitive molecular indicators linking maternal oxycodone use to disrupted fetal cardiovascular, metabolic, and neurodevelopmental pathways. This study provides a novel systems-level framework for understanding opioid-induced placental signaling alterations and lays the groundwork for developing EV-based diagnostic biomarkers and targeted interventions.

Extracellular vesicles (EVs) are heterogeneous, lipid bilayer-enclosed vesicles secreted by cells. Research on EVs dates back to the 1940s, and the term “exosomes” - a major subtype of EVs - was coined in 1981 to describe small membrane vesicles shed from cells. However, it is only in the past two decades that research in this area has expanded rapidly. By transferring functional biomolecules, EVs play a pivotal role in intercellular communication and regulate a wide range of cellular functions under both physiological and pathological conditions. Owing to their high biocompatibility, capacity to protect encapsulated cargo from degradation, and ability to cross biological barriers, EVs also show great promise as biomarkers and drug-delivery systems. Following the first, albeit unintentional, isolation of EVs in 1946, the 80th anniversary of EV research is now approaching. In this review, we trace the history of EV research and summarize key advances in the field. We also discuss current challenges and future prospects in this rapidly evolving area.

Aim: This study aimed to investigate whether vesicle-like nanoparticles derived from the GeGen decoction (GGD-PDVLNs) represent a key bioactive component responsible for its anti-colitis effects and to elucidate their underlying mechanisms, particularly focusing on gut microbiota modulation.

Methods: The GeGen decoction (GGD) was subjected to differential centrifugation following boiling, yielding vesicle-like nanoparticles. Structural analysis confirmed that these nanoparticles have a lipid bilayer and can resist digestion by simulated gastrointestinal fluids. These nanoparticles were administered orally to mice with chronic colitis induced by dextran sulfate sodium to evaluate their therapeutic effects.

Results: GGD-PDVLNs effectively mitigated intestinal inflammation by reducing the secretion of pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, tumor necrosis factor-alpha (TNF-α)], elevating levels of the anti-inflammatory cytokine IL-10, alleviating intestinal damage, and enhancing intestinal barrier function, all while exhibiting a favorable biosafety profile. Notably, their therapeutic action depended on gut microbiota modulation. GGD-PDVLNs restored microbial homeostasis, increased microbial diversity, and enriched probiotic populations. In pseudo-germ-free mice, GGD-PDVLNs lost efficacy, confirming microbiota-dependent mechanisms.

Conclusion: Vesicle-like nanoparticles are an important active component of GGD. Our findings demonstrate that GGD-PDVLNs significantly ameliorate colonic inflammation through microbiota-dependent mechanisms.

Spinal cord injury (SCI) is a highly disabling disorder of the central nervous system for which no curative therapy is currently available. In recent years, extracellular vesicles - particularly exosomes - have been investigated as cell-free therapeutic approaches in experimental models, owing to their low immunogenicity, favorable biocompatibility and capacity to traverse the blood-spinal cord barrier under specific conditions or delivery routes. This Review summarizes the therapeutic activities and mechanisms of exosomes from diverse sources - including mesenchymal stem cells, immune cells and neural cells - in SCI repair. Reported mechanisms include modulation of the inflammatory microenvironment; inhibition of apoptosis and pyroptosis; mitigation of ferroptosis; promotion of angiogenesis and axonal regeneration; and restriction of glial scar formation. We also discuss advances aimed at enhancing exosome efficacy through cell preconditioning, engineering strategies and integration with biomaterials. Although exosome-based approaches are promising, challenges remain in standardization, targeted delivery and long-term safety. Future work should elucidate the underlying mechanisms and advance clinical translation to robustly evaluate the therapeutic potential of exosomes for SCI repair.

Extracellular vesicles (EVs) are emerging as promising tools for regenerative medicine and drug delivery, offering unique therapeutic advantages. However, their clinical translation - in Australia and globally - faces persistent challenges. These are commonly framed as technical issues stemming from inherent EV variability and the absence of standardized potency assays. While no EV-based therapeutic has yet received full market approval from any major regulatory agency, this Perspective argues that the barriers to translation are not solely technical but reflect limitations within current regulatory frameworks. In Australia, the Therapeutic Goods Administration (TGA) requires biological products to be included on the Australian Register of Therapeutic Goods (ARTG) before supply to market. However, several alternative regulatory pathways exist that can facilitate clinical access to “unapproved” products. Through analysis of these pathways, and comparison with international approaches, this Perspective highlights how regulatory inflexibility may be as significant a barrier to translating EV medicines as the technical difficulties themselves. Drawing on insights from governmental inquiries into the approval and subsidization of emerging medicines, the Perspective calls for reform of Australia’s regulatory systems - including development of EV-specific guidance and policy that introduces adaptive assessment pathways - to better support the safe and timely integration of novel biotechnologies.

Aim: To compare specific functional features of equine amniotic mesenchymal cells (eAMCs) and their extracellular vesicles (EVs) through proteomic analysis.

Methods: eAMCs were obtained by enzymatic digestion and their EVs were isolated by ultracentrifugation. Cells and EVs were characterized according to ISCT and MISEV guidelines. A proteomic analysis of both eAMCs and EVs was conducted. The raw data files were analyzed using FragPipe 22 and uniprotkb equus_caballus_reviewed database (20.02.2025) to obtain protein identifications (false discovery rate = 0.01) and their respective label-free quantification values using recommended parameters. Statistical analysis was performed based on the combined_protein.tsv file using FragPipe-Analyst. A cutoff of the adjusted P-value of 0.05 along with a |log2 fold change| of 1 has been applied to determine differentially expressed proteins in the comparison.

Results: A total of 3,631 proteins were identified, of which 3,147 were identified with more than two peptides. Among these, 2,235 were exclusive to eAMCs, 697 were shared between eAMCs and EVs, and 71 were exclusive to EVs. eAMCs and EVs revealed distinguished proteomic profiles, differentially expressing proteins involved in biological processes related to tissue regeneration. Proteins promoting anti-inflammatory activity, oxidative stress resistance and angiogenesis exhibited increased expression levels in eAMCs, while extracellular matrix organization and deposition were predominantly upregulated in EVs.

Conclusions: Equine eAMCs and EVs are characterized by a distinct proteomic profile showing the expression of different sets of proteins involved in regenerative processes and thus different therapeutic properties. This also highlights their coordinated activity on tissue homeostasis and regeneration mechanisms.

Aim: Acute pancreatitis (AP) is an inflammatory disorder of the pancreas lacking specific therapy and frequently complicated by oxidative stress (OS) and long-term endocrine dysfunction, including pancreatogenic diabetes. Pomegranate-derived nanovesicles (PgNVs) contain bioactive lipids, proteins, and metabolites responsible for many health benefits of pomegranate juice. This study evaluated whether prophylactic PgNVs administration could mitigate pancreatic injury, OS, systemic inflammation, and subsequent endocrine impairment in a murine model of severe L-arginine-induced AP.

Methods: Male C57BL/6J mice were distributed to three groups: control, AP, and AP + PgNVs. A single subcutaneous dose of PgNVs (10 µg; ≈ 5 × 10¹⁰ particles) was administered 2 h before AP induction via intraperitoneal L-arginine. Animals were analyzed at 3, 7, 30, and 60 days post-induction. PgNVs were isolated using differential centrifugation, tangential-flow filtration, and size-exclusion chromatography.

Results:PgNVs pretreatment preserved pancreatic architecture, reduced edema, amylase activity, and interleukin 6 (IL-6) levels in both tissue and plasma by limiting nuclear factor-κB-p65 phosphorylation. PgNVs restored redox balance by upregulating NQO1 [NAD(P)H quinone dehydrogenase 1], improving oxidized glutathione (GSSG)/reduced glutathione (GSH) and homocystine/homocysteine ratios, and maintaining PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) expression. PgNVs protected β-cell mass, enhanced insulin secretion, and normalized glucose tolerance after AP. Proteomic profiling revealed that PgNVs changed extracellular vesicle composition, lowering pro-inflammatory markers [fibronectin, dipeptidyl peptidase-4 (DPP4)] and restoring anti-inflammatory ITIH4 (inter-alpha-trypsin inhibitor protein heavy chain 4).

Conclusion:PgNVs exert anti-inflammatory and antioxidant protection in experimental AP and preserve long-term endocrine function, highlighting their potential as a natural nanotherapeutic strategy to prevent pancreatic injury and post-pancreatitis diabetes.

In situ tissue engineering, which activates the body’s innate regenerative capacity, has demonstrated superior clinical translation potential than traditional ex situ approaches. Small extracellular vesicles (sEVs), as natural nanovesicles, can excellently mimic the paracrine functions of cells and are thus emerging as promising cell-free alternatives for in situ tissue engineering. Despite advantages such as low immunogenicity, multi-target regulatory capabilities, and cross biological barriers availability, the therapeutic sustainability of sEVs is limited by their rapid clearance in vivo, underscoring the need for effective delivery systems. This review systematically summarizes the sources and bioactivities of sEVs, delineates the design principles and technological advances in sEVs delivery systems, and highlights their application in tissue engineering, while also outlining future trajectories for the development of intelligent delivery platforms.

Aim: To evaluate the therapeutic efficacy and mechanisms of tumor necrosis factor-alpha (TNF-α) preconditioned mesenchymal stem cells (MSCs)-derived small extracellular vesicles (hereafter abbreviated as T-sEV), along with the control small extracellular vesicles (sEV, representing vesicles from naive/unstimulated MSCs), in mitigating inflammatory retinal injury.

Methods: T-sEV were isolated from TNF-α-preconditioned MSCs and systematically characterized. Small RNA sequencing was performed to identify the microRNA (miRNA) cargo of T-sEV. The effect of T-sEV on lipopolysaccharide (LPS)-induced M1 macrophage polarization was assessed by flow cytometry. Integrated bioinformatic analysis linked T-sEV miRNAs to macrophage transcriptome changes. T-sEV were administered intravitreally in a murine sodium iodate (NaIO₃)-induced retinal degeneration model. Electroretinography (ERG), optical coherence tomography (OCT), flow cytometry, intraocular pressure (IOP) and systemic examinations were conducted.

Results: T-sEV exhibited an enrichment of anti-inflammatory miRNAs, notably miR-146a-5p. They were efficiently internalized by macrophages, significantly suppressing M1 polarization, as evidenced by the decreased percentage of cluster of differentiation (CD)11b⁺/CD86⁺ cells (29.60% ± 2.30%) compared to the sEV group (34.90% ± 1.57%, P < 0.05). Analysis showed T-sEV miRNAs targeted and downregulated key pro-inflammatory genes such as Cd86 and Il1r1. In vivo, T-sEV treatment significantly preserved retinal a- and b-wave amplitudes and structural integrity. T-sEV treatment markedly reduced retinal macrophage infiltration, decreasing the proportion of F4/80⁺CD11b⁺ cells to 0.38% ± 0.13%, significantly lower than in the sEV group (1.66% ± 0.47%, P < 0.01). No adverse effects on IOP or systemic markers were observed.

Conclusion: TNF-α preconditioning enhances MSC-derived sEV therapeutic capacity by enriching their anti-inflammatory miRNAs. T-sEV suppress pro-inflammatory macrophage activation and provide superior neuroprotection in a retinal degeneration model, indicating a safe and promising cell-free therapeutic strategy.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide, characterized by progressive cognitive decline and a current lack of effective curative treatments. In recent years, extracellular vesicle (EV) therapies have emerged as a cutting-edge approach in AD research due to their unique biological properties. This review systematically examines the role of EVs in the pathogenesis of AD, including their cellular origins and functional attributes. We detail the molecular mechanisms mediated by EVs that influence AD progression and summarize the latest advancements in EV-based therapeutic strategies. Additionally, the review addresses the challenges faced in translating these therapies from bench to bedside, such as standardization, delivery optimization, and safety concerns. Future research directions are discussed to foster the development of innovative and effective treatments for AD. By providing a comprehensive overview, this article aims to lay a theoretical foundation and offer valuable insights for the advancement of novel therapeutic interventions targeting AD.

Aim: Extracellular vesicles (EVs) released by mesenchymal stem cells (MSCs), known as MSC-EVs, have gained attention as potential treatments owing to their immunomodulatory functions. Despite growing clinical interest, donor-to-donor inconsistencies remain key challenges for standardizing MSC-EV production under good manufacturing practice (GMP) conditions. This work aimed to systematically compare the molecular and functional consistency of EVs derived from three independent human adipose tissue-MSC donors.

Methods: GMP-grade EVs were initially isolated using tangential flow filtration on a large scale and then characterized by multiple biophysical analyses. To characterize the protein composition of EVs across batches, quantitative proteomic analysis was performed using tandem mass tags and mass spectrometry. For functional validation, an in vitro macrophage inflammation assay was conducted by treating natural lipopolysaccharide-stimulated cells with EVs, and cytokine levels were measured using enzyme-linked immunosorbent assays (ELISA).

Results: Quantitative proteomic profiling identified 2,615 proteins, of which 84%-94% were not significantly changed across batches, highlighting a robust core proteome. Notably, 361 membrane-associated proteins were consistently conserved, including transporters, adhesion molecules, and signaling receptors, implicating these components in EV-mediated intercellular communication and immunomodulation. Functional analysis using an in vitro macrophage inflammation model demonstrated that all EV batches reproducibly suppressed pro-inflammatory cytokine production in a dose-dependent manner, with no significant inter-batch differences.

Conclusion: Collectively, these findings indicate that MSC-EVs maintain both molecular and functional stability across different donors, and that a conserved proteomic signature underlies their reproducible anti-inflammatory activity. This study provides a foundation for establishing standardized quality criteria and advancing MSC-EVs toward clinical therapeutic applications.

Plant-derived exosome-like nanovesicles (PELNs), as an emerging “green” nanoplatform, exhibit broad pharmacological activities, low immunogenicity, and inherent advantages as natural drug carriers. They show great potential in the pharmaceutical, cosmetic, and health supplement sectors. The clinical application of PELNs is heavily contingent on their safety profile, which is intricately linked to the administration route. This opinion compares the safety implications of the two primary routes: oral administration versus intravenous injection. Current evidence indicates that intravenous administration of PELNs triggers complement activation, immune responses, and hepatorenal toxicity; even surface engineering modifications cannot completely eliminate these risks. In contrast, oral administration of PELNs may achieve superior safety by leveraging the gastrointestinal tract’s ability to effectively reduce the immunogenic components. Based on these findings, we advocate for the prioritization of oral delivery in the future development of PELNs, given its superior safety profile for realizing their potential as natural therapeutics and drug delivery systems.

Aim: Extracellular vesicles (EVs) are emerging mediators of intercellular communication capable of transporting nucleic acids, including plasmid and genomic DNA. This study aimed to investigate the presence, enrichment, and protection of distinct DNA classes in EV-enriched fractions from Saccharomyces cerevisiae (S. cerevisiae).

Methods: Two plasmids were analyzed: the high-copy YEp352 (2µ) and the centromeric pRS316. EV-enriched fractions were isolated from yeast culture supernatants, and the presence of plasmid, ribosomal [18S ribosomal DNA (RDN18)], and mitochondrial [cytochrome c oxidase subunit I (COX1)] DNA was assessed. DNase digestion assays were performed to evaluate DNA protection, and transformation assays were conducted using S. cerevisiae and Escherichia coli. Enrichment of endogenous vs. exogenously added plasmid DNA was also compared.

Results: Both plasmids were strongly enriched in EV fractions relative to culture supernatants. However, only pRS316 exhibited partial resistance to DNase digestion, suggesting encapsulation, while YEp352 remained fully susceptible. Despite this protection, pRS316-associated EVs did not mediate transformation of yeast or bacterial recipients, indicating compromised integrity or inefficient delivery. Endogenously produced plasmid DNA showed significantly higher enrichment than exogenously added DNA. Ribosomal and mitochondrial DNA were also detectable in EVs but were highly sensitive to DNase degradation, indicating minimal protection.

Conclusion: These findings demonstrate that selective DNA incorporation into EVs depends on both DNA type and intrinsic plasmid features. The results suggest that plasmid properties influence EV-mediated transport and protection, highlighting the selective and cargo-specific nature of DNA packaging in yeast EVs.