The central innovation rests on a pair of molecular tools called split inteins. Inteins are self-splicing protein elements that can excise themselves from a larger protein while stitching the flanking fragments back together with a peptide bond. When an intein is split into two non-functional halves — an N-terminal fragment and a C-terminal fragment — the two halves can reassociate spontaneously and reconstitute splicing activity without any external catalyst. The researchers exploited the well-characterized Npu split intein pair, fusing the N-terminal component, NpuN, to CD9, a tetraspanin membrane protein that is abundantly sorted into extracellular vesicles, and fusing the C-terminal component, NpuC, to the protein cargo of interest. The logic is elegant: because CD9 is naturally enriched on vesicle membranes, any cargo tethered to it through the reconstituted NpuN/NpuC interaction should be dragged along into the vesicle lumen or membrane during vesicle biogenesis.

What distinguishes this design from earlier intein-based loading schemes is the placement of a pH-sensitive linker between NpuC and the cargo. The researchers reasoned that this sequence would remain intact at neutral pH but would cleave under acidic conditions — precisely the environment found inside the intraluminal vesicles of producing cells and within the endosomes of recipient cells after vesicle uptake. In principle, this means the cargo is carried into vesicles while attached, then released from its membrane anchor when the vesicle interior or the recipient endosome acidifies, freeing the protein to diffuse into the cytosol and perform its function rather than remaining stuck to the vesicle membrane.

To test whether the system actually improves loading, the team fused two fluorescent reporter proteins, DsRed2 and tdTomato, to NpuC and compared engineered vesicles against vesicles loaded by passive means. The split-intein approach improved the loading of both reporters by approximately 1.3-fold relative to passive loading. That figure is modest rather than dramatic, but it represents a consistent, reproducible gain achieved with a single genetic construct and no chemical transfection reagents, electroporation, or viral envelope proteins — all of which complicate conventional loading workflows and raise safety or manufacturing concerns for clinical translation.

The most consequential demonstration, however, came when the researchers swapped the fluorescent reporters for a genuinely functional enzyme: the piggyBac transposase. This enzyme recognizes specific inverted terminal repeat sequences in DNA and excises any genetic material flanked by them, a property widely used in genome engineering. The team engineered donor cells to produce vesicles carrying NpuC-fused piggyBac transposase, isolated those vesicles, and added them to reporter cells whose genomes contained a 5′ITR–EGFP–polyadenylation signal–3′ITR cassette positioned so that its excision would switch on expression of mCherry. When the vesicles were taken up, the transposase excised the flanked cassette and the reporter cells turned red — direct evidence that a catalytically active enzyme had been loaded into vesicles, delivered across the recipient cell membrane, released into the cytosol, and had performed a genome-modifying reaction.

This functional delivery assay matters because most cargo-loading studies stop at showing that a fluorescent protein is present inside vesicles. Fluorescence can be detected without the protein being folded correctly, enzymatically competent, or accessible to the recipient cell’s cytoplasm. The piggyBac experiment closes that gap: excision of a genomic cassette is an all-or-nothing molecular event that cannot occur unless the transposase arrives intact and active. It also showcases a potential application beyond delivery for its own sake — vesicle-borne transposases could, in future work, be used to remove or rearrange specific genetic elements in living cells without exposing them to viral vectors.

A notable design choice is the deliberate avoidance of VSV-G, the vesicular stomatitis virus glycoprotein that is commonly used to pseudotype extracellular vesicles and enhance their fusion with target cell membranes. VSV-G pseudotyping improves delivery efficiency but imports a viral envelope protein into the system, which can alter vesicle tropism, trigger immune responses, and blur the line between a genuinely extracellular-vesicle-based therapy and a disguised viral vector. By building a loading and delivery system that works entirely with endogenous vesicle components — CD9 as the membrane anchor and the split intein as the molecular glue — the authors offer a platform that keeps the vesicles closer to their natural composition.

The characterization of the engineered vesicles relied on transmission electron microscopy to confirm vesicle morphology, dynamic light scattering to measure size distributions, and dot blot detection to verify the presence of the engineered mEmerald-CD9 membrane protein and the DsRed2 cargo in the vesicle fraction. These are standard and appropriate first-line techniques for confirming that the particles being studied are indeed membrane-bound vesicles of the expected size range and that the engineered constructs are physically associated with them.

The authors are candid about an important limitation: no endogenous extracellular vesicle markers or negative contamination markers were assessed in this study, which means vesicle identity and sample purity were not established by protein marker profiling. In the extracellular vesicle field, where preparations can be contaminated with protein aggregates, lipoproteins, and other non-vesicular particles, marker-based validation according to community guidelines is considered essential for attributing observed effects unambiguously to vesicles themselves. The functional piggyBac result strongly suggests that active enzyme was delivered in a membrane-associated form, but rigorous marker profiling will be needed before the platform’s mechanism of uptake and release can be stated with full confidence.

Even with those caveats, the study adds a genuinely useful tool to the extracellular vesicle engineering toolbox. The split-intein system is modular — any protein of interest can, in principle, be fused to NpuC — and it operates in a single step during natural vesicle production, avoiding post-isolation manipulation that can damage vesicles. The pH-sensitive release element adds a built-in trigger keyed to the acidic compartments that vesicles naturally encounter after uptake. If follow-up work confirms marker-defined purity, optimizes the 1.3-fold loading gain, and extends the functional delivery results to additional enzyme classes and primary cells, the approach could help extracellular vesicles move closer to their long-promised role as programmable, virus-free carriers of therapeutic proteins.

Subject of Research: Split-intein-based engineering of extracellular vesicles for protein cargo loading and functional delivery

Article Title: Split-intein-mediated engineering of VSV-G-free extracellular vesicles for enhanced cargo loading and delivery of functional proteins

Article References: Ghazi Birjandi, R., & Dehghani, H. (2026). Split-intein-mediated engineering of VSV-G-free extracellular vesicles for enhanced cargo loading and delivery of functional proteins. Scientific Reports. https://doi.org/10.1038/s41598-026-75437-9

Image Credits: AI Generated

DOI: 10.1038/s41598-026-75437-9

Keywords: extracellular vesicles, split inteins, cargo loading, CD9, tetraspanins, protein delivery, piggyBac transposase, VSV-G-free, pH-sensitive linker, nanocarriers, Scientific Reports, vesicle engineering

The case for treating large extracellular vesicles as their own category rests on a fundamental difference in origin. Small extracellular vesicles arise from two well-characterised sources: the endosomal system, which produces exosomes when multivesicular bodies fuse with the plasma membrane, and the plasma membrane itself, which buds off small ectosomes. Most large extracellular vesicles, by contrast, stem directly from the plasma membrane, although the review emphasises that the various subtypes follow strikingly different biogenesis routes. This distinction matters because it shapes what the vesicles carry, how they are released, and what they do once they arrive at a recipient cell. A vesicle born from a dying cell’s collapsing membrane carries a very different cargo than one pinched off from the rear of a migrating cell or extruded from a neuron under proteotoxic stress.

The classical cast of large vesicles includes apoptotic bodies, microvesicles and large oncosomes, each recognised for years and each associated with distinct biology. Apoptotic bodies form as dying cells disassemble themselves, a process driven by caspase-mediated activation of ROCK1, which triggers the actomyosin contraction underlying membrane blebbing. These fragments are far more than debris: they broadcast find-me and eat-me signals that orchestrate the orderly clearance of dead cells by phagocytes, and recent work shows they can stimulate stem cell proliferation during tissue maintenance and couple bone resorption to bone formation. Microvesicles, shed directly from the plasma membrane of viable cells, depend on the ARF6–myosin light chain–actomyosin axis for their scission, and tumour cells exploit this pathway to spread oncogenic receptors such as EGFRvIII and drug-resistance proteins like P-glycoprotein to neighbouring cells. Large oncosomes, the giant vesicles shed by cancer cells, have been linked to loss of the formin DIAPH3 and to the reprogramming of fibroblasts in the tumour microenvironment through MYC-dependent mechanisms.

The most exciting chapters of the review concern the newer subtypes, several of which were unknown when the extracellular vesicle field consolidated its nomenclature under the MISEV guidelines. Migrasomes, discovered in 2015 by Li Yu’s group, form on retraction fibres left behind by migrating cells and serve as organelles for controlled release of cytoplasmic contents during cell migration. Their formation depends on the assembly of micron-scale tetraspanin-enriched macrodomains, driven by sphingomyelin synthase 2, phosphatidylinositol 4,5-bisphosphate signalling through Rab35, and palmitoylation of the tetraspanin Tspan4. Migrasomes have since been implicated in zebrafish organ morphogenesis, embryonic angiogenesis, mitochondrial quality control through a process termed mitocytosis, and even coagulation, with neutrophil-derived migrasomes identified as essential components of the clotting system.

Equally remarkable are the vesicles that cells use to jettison damaged components. Exophers, first described in Caenorhabditis elegans neurons in 2017, are enormous vesicles through which stressed neurons extrude toxic protein aggregates and dysfunctional mitochondria, providing a proteostasis escape route with obvious implications for neurodegenerative disease. Mitophers, reported in 2023, represent a form of mitochondria-specific ectocytosis through which sperm cells regulate their mitochondrial complement and, consequently, their fertility, establishing a paradigm for cargo-selective packaging of entire organelles into large vesicles. Blebbisomes, characterised in 2025, are large, organelle-rich vesicles with startling cell-like properties, carrying broad organellar cargo and expanding the functional repertoire attributed to vesicle-mediated communication. Large ageing neutrophil-derived vesicles, or LAND-Vs, add an anti-inflammatory dimension: they control complement activation and facilitate the resolution of inflammation, revealing that large vesicles can dampen immune responses as well as provoke them.

Beneath this diversity lies a set of shared physical and biochemical principles that the review dissects with technical precision. Membrane budding and scission require the coordination of actomyosin contractility, hydrostatic pressure and lipid remodelling. The transbilayer distribution of phospholipids emerges as a recurring regulatory node: flippases maintain lipid asymmetry, while scramblases such as TMEM16F collapse it in a calcium-dependent manner, exposing phosphatidylserine on the outer leaflet and promoting both vesicle release and the engulfment signals that mark apoptotic material for clearance. Disruption of flippase localisation in C. elegans increases extracellular vesicle budding, demonstrating that lipid asymmetry actively suppresses vesiculation in healthy membranes. Protein crowding at the membrane, tetraspanin microdomains, lipid rafts and caveolin-dependent dynamics each contribute to curvature generation at specific sites, while the ESCRT machinery contributes its membrane-scission capabilities to several of these pathways.

Cargo sorting into large vesicles follows equally mechanistic rules. The ARF6–Exportin-5 axis delivers pre-microRNA cargo into tumour microvesicles, Cdc42 acts as a regulatory node for microvesicle biogenesis, and RNA-binding proteins govern which transcripts are packaged. Zipcode-like sequence elements enrich specific mRNAs in microvesicles, and integrin pairing with extracellular matrix proteins determines where migrasomes form on retraction fibres. Delivery to recipient cells proceeds through clathrin-independent endocytosis and macropinocytosis, although the review is careful to note that the field still lacks definitive experimental support for functional cargo transfer in many contexts, a caution that reflects growing scrutiny of the extracellular vesicle cargo-transfer hypothesis.

The pathological implications are broad and, in several cases, already translating toward clinical applications. In cancer, large vesicles transfer oncogenic receptors, promote angiogenesis through CD147 and VEGF signalling, carry retrotransposon elements and amplified oncogene sequences, and mediate immune evasion through vesicle-associated PD-L1, which Epstein–Barr virus LMP1 enhances. In neurodegeneration, microglia convert aggregated amyloid-beta into neurotoxic forms through microvesicle shedding, while macrophage-derived migrasomes activate complement-dependent blood–brain barrier damage in cerebral amyloid angiopathy and promote paracrine senescence in ageing brain. Inflammation, thrombosis and kidney injury each feature large-vesicle mechanisms, with urinary podocyte-derived migrasomes emerging as early indicators of glomerular damage and plasma apoptotic bodies serving as non-invasive biomarkers of apoptosis in ischemic stroke.

Methodological rigour occupies a central place in the review’s argument. The authors catalogue the technologies now enabling multidimensional characterisation, from cryo-electron microscopy and super-resolution structured illumination microscopy to nanoparticle tracking analysis, tunable resistive pulse sensing, asymmetric flow field-flow fractionation and microfluidic affinity capture. Deep-learning platforms for automated detection of vesicle exocytosis and self-supervised image enhancement for long-term subcellular recording are extending what live imaging can reveal. Yet the review is equally candid about pitfalls: fluorescent lipophilic dyes can confound uptake studies, isolation methods such as ultracentrifugation and tangential flow filtration yield different preparations, and loss-of-function studies and mechanistic analyses must be appropriately integrated to ensure rigour and reproducibility. Engineered migrasomes that serve as thermally stable vaccination platforms hint at the therapeutic horizon, but the authors stress that biodistribution depends on cell source, administration route and targeting ligands, and that the field’s progress will depend on pairing new tools with the disciplined standards set out in the MISEV2023 framework. What emerges is a portrait of a maturing discipline: one in which the largest and most structurally complex members of the extracellular vesicle family are finally being studied with the systematic attention their biology demands.

Subject of Research: Subtypes, biogenesis and functions of large extracellular vesicles

Article Title: Towards a systematic understanding of the diverse subtypes, biogenesis and functions of large extracellular vesicles

Article References: Huang, Y., Xie, R., Ma, J., & Yu, L. (2026). Towards a systematic understanding of the diverse subtypes, biogenesis and functions of large extracellular vesicles. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/s41580-026-01021-8

Image Credits: AI Generated

DOI: 10.1038/s41580-026-01021-8

Keywords: large extracellular vesicles, migrasomes, apoptotic bodies, microvesicles, large oncosomes, exophers, blebbisomes, mitophers, plasma membrane budding, tetraspanins, extracellular vesicle biogenesis, cell-cell communication