Gene editing tools such as ZFN, TALEN and CRISPR-Cas9 have broadened targeted genome modification across many species and genes. However, ZFNs and TALENs are relatively complex and costly to construct. CRISPR-Cas9 is easier to use but off-target effects remain a concern. Combining gene editing with EV modification enables various modes of editing including single base substitution^[55], insertion or deletion of specific base sequences^[56], translational editing^[57], and combined inactivation or activation of multiple targets^[58,59] can be achieved. This combination can not only regulate the cargo and surface-modifying molecules of vesicles but also modify the overall metabolic performance of cells, ultimately enhancing the production of high-quality extracellular vesicles. Nevertheless, this field is still at an early stage with challenges, such as how to increase the yield and purity of EVs how to ensure the safety and effectiveness of gene editing tools, and how to achieve precise delivery and regulation in vivo.

However, the development prospects in this field remain very broad. Zanella and colleagues constructed a BL21(DE3)Δ60 engineered strain through CRISPR/Cas9-based genome editing, which improved both recombinant protein expression and OMVs loading efficiency^[60]. In archaea, CRISPR-Cas9 gene editing of methanogenic archaea was reported for the first time in 2023, and targeted knockout of Msv_1527 genes resulted in a 100% increase in yield of EVs^[61]. In fungal systems, a specific CRISPR-Cas9 editing platform inhibited PMA1 gene expression in Candida albicans and significantly reduced PMA1 protein loading in EVs, providing a new technical path for targeted therapy of inflammatory bowel disease^[62]. These research results not only establish a precise regulation system for the production and function of EVs, but also open new research directions for microbial engineering in biomedicine.

Breakthrough advances in synthetic biology have significantly enhanced humanity’s ability to rationally design and selectively modify life systems. In microbial genomic engineering, research teams have successively achieved whole genome synthesis technologies for both prokaryotes and eukaryotes. They have verified the biological functional integrity of synthetic life systems through artificial genome reconstruction. Notably, artificial chromosome reconstruction technology has shown strong genetic reshaping potential, with its dynamic recombination characteristics providing a new regulatory dimension for the directed evolution of cellular metabolic networks^[63,64]. Relying on the genomic synthesis technology platform, researchers can systematically reshape the biosynthetic pathways of EVs by optimizing the protein expression regulatory network and constructing a framework for the sequential expression of non-coding RNAs, thereby establishing an intelligent vesicle generation system. This innovative approach, which integrates synthetic biology with systems biology, will effectively shorten the development cycle of engineered strains with specific industrial characteristics.

For the E. coli Nissle 1917 (EcN) chassis system, researchers have used genome streamlining engineering to delete non-essential gene clusters. This approach has significantly improved the biosynthesis efficiency of OMVs^[65]. Fusion antigen of influenza virus, human papillomavirus, pneumococcus, Staphylococcus aureus, and Acinetobacter baumannii displayed on OMVs produced by E. coli through genetic engineering can stimulate the body to produce specific antibodies against these pathogenic microorganisms, thereby playing an effective preventive role^[66,67]. These results fully demonstrate the multifunctional application value of genome editing technology in the engineering transformation of MEVs, which not only optimizes the yield of EVs, but also provides important technical support in terms of immunogenicity enhancement and regulation of physical properties.

Beyond direct genomic modifications of engineered bacteria, various engineered bacteria can now be designed and constructed. This enables the synthesis and release of vesicles in vivo, facilitating biomedical applications such as disease prevention and proactive health interventions. Zhou et al. developed an orally administrable engineered probiotic that overexpresses catalase and superoxide dismutase to eliminate reactive oxygen species, thereby improving the treatment of intestinal inflammation^[68]. The “protective suit” system for live bacteria established by Liu shows promise in providing new strategies for bacterial transplantation and prevention of enteritis^[69]. Cui et al. have been conducting long-term research on optogenetic engineered intestinal bacteria as living drugs^[70]. By empowering engineered bacteria with synthetic biology techniques, they have expanded the applications of these bacteria in areas such as enteritis, gut-brain axis regulation, gut-kidney axis regulation, and blood glucose control^[71-73]. The customization, efficacy enhancement, and safety control modifications of engineered bacteria and their EVs will provide robust support for the biomedical applications of in situ synthesized EVs in vivo.

This study summarizes some strategies and effects of using genetic engineering methods to modify chassis cell genes [Table 2].

In recent years, the rapid development of computational biology and AI in protein science has provided powerful tools for the rational design of membrane proteins. Advanced protein structure/function prediction tools such as AlphaFold^[84], protein large language models^[85], and others (e.g., Rosetta, RFdiffusion^[86]) have enabled researchers to more accurately predict and design novel membrane proteins, even achieving complete de novo design.

These techniques have been successfully applied to several OMV related research cases, including the application of AlphaDesign to design in vivo active inhibitors of the phage defense protein RcaT-Sen2^[87]. In membrane fusion mechanism innovation, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) like spike proteintransmembrane domain designed based on the evolutionary scale modeling 2 (ESM 2) language model enables OMV to achieve efficient membrane fusion with angiotensin-converting enzyme 2 (ACE2) expressing cells^[88]. Functionalized OMVs have also shown great potential in cancer diagnosis and treatment. The antimicrobial peptide Polybia-mastoparan I (MPI) loaded in E. coli OMV provides a novel delivery vehicle for bladder cancer treatment^[89]; The fusion expression of pH-sensitive green fluorescent protein and EVs membrane proteins such as CD63 enables EVs to achieve fluorescence signal activation in the acidic tumor microenvironment, providing a real time visualization tool for cancer diagnosis and treatment monitoring^[90]. In the future, with the development of multimodal AI protein design tools, artificial design of vesicle surface proteins will achieve higher precision functional programming, promoting the application of OMVs in precision medicine. These breakthrough advances not only demonstrate the synergistic effects of computational biology, protein engineering, and nanotechnology but also lay a solid technical foundation for the intelligent design and clinical translation of OMVs.

In protein structure prediction, elucidation and specific molecular design, several recent studies have promoted breakthroughs in the biomedical application of OMVs through the integration of computational biology, synthetic biology, and nanobiotechnology. Researcher used synthetic biology technology to construct recombinant probiotics and modify membrane components to display BMP-2 and CXCR4 on the surface of bacterial extracellular vesicles (BEVs), achieving dual functions of bone targeting and bone regeneration, thereby providing an innovative treatment for osteoporosis^[91]. Other researchers engineered EVs by fusing HaloTag with vesicle anchoring proteins on the EV surface, enabling the targeted display of molecules such as GalNAc to successfully target human liver cells^[92].

Moreover, the integration of optogenetics technology and bioelectronic medicine has opened up new paths for disease diagnosis and treatment. Optical imaging detection and bioelectronic drugs constructed using light-controlled engineered bacteria have enabled precise monitoring and dynamic intervention for chronic diseases including kidney disease^[72]. In tumor immunotherapy, two innovative strategies demonstrate the unique advantages of OMVs as nanoscale immune adjuvants. Firstly, OMV surface nanobodies serve as biological scaffolds that activate immune cells through specific binding to tumor-associated antigens, providing a targeted immune activation strategy for solid tumor treatment^[93]. Secondly, engineered photosynthetic bacteria and their OMVs are designed as intelligent antigen capture systems, capable of efficiently enriching tumor antigens and delivering them to the tumor-draining lymph node (TDLN) region, thereby enhancing the body’s anti-tumor immune response^[94]. Together, these studies form a complete technological chain - from protein structure prediction to functionalized vector design, and then to precise disease intervention. This work deepens our understanding of the biophysical properties of OMVs and provides theoretical support and technical paradigms for developing next-generation intelligent biologics.

Using synthetic biology methods, researchers have achieved multilevel optimization design. Precise regulation of the spatial arrangement of 5'UTR, 3'UTR and IRES elements has increased target protein expression levels by thousands of times. Polycistronic mRNA technology enables a single mRNA molecule to encode multiple functional proteins, such as components of the CRISPR-Cas9 system. Specific RNA secondary structures can enhance miRNA binding capacity, achieving efficient gene regulation. These strategies provide important technical support for the functional modification of EVs.

Cui et al. proposed an economical and efficient technology to prepare engineered OMVs by overexpressing pre-miRNA in E. coli^[95]. Similarly, Santos et al. demonstrated that exogenous loading of miR-195-5p, a tumor suppressor miRNA, into EVs enhances its anti-tumor activity and improves targeted therapy response in melanoma against patients with B-raf proto-oncogene mutations^[96]. This finding highlights the potential of EV based strategies to enhance clinical outcomes. These studies indicate that through genetic modification and physical loading methods, OMVs can be designed as efficient anticancer carriers, providing new ideas and technical means for developing novel cancer treatments. However, issues such as delivery efficiency, functional stability, immunogenicity, and production costs still need to be addressed further to realize their clinical translation potential.

Mammalian cells contain over 500 types of RNA binding proteins (RBPs), providing a rich source of natural regulatory elements for synthetic biology^[97]. Bioengineered, enhanced EVs can deliver collagen mRNA into the skin, promoting the production of collagen lost due to aging in recipient skin cells^[98]. For surface functionalization, the fusion system of the bacterial membrane protein ClyA and SnoopCatcher enables the modular capture of vesicle surface proteins, and this strategy, combined with intestinal in situ synthesis technology, can dynamically release engineered EVs in the tumor microenvironment to enhance antitumor activity^[99,100]. Zhuang and colleagues further crossed species boundaries by constructing photodynamic hybrid vesicles (BPNs) that fuse plant thylakoid membranes with bacterial OMVs. These vesicles generate reactive oxygen species to directly ablate tumor tissues under laser irradiation^[101]. In addition, a synergistic strategy of chemical modification and bioengineering showed unique advantages: Shen et al. integrated the tyrosine rich protein statherin into the surface of OMVs, and significantly extended its blood circulation time and increased tumor accumulation rate through polyethylene glycol modification^[102]. These multidimensional engineering strategies reveal the molecular design potential of EVs as “programmable biological carriers”. They also provide an innovative technical paradigm for the integration of drug delivery, immune regulation, and real-time diagnosis and treatment in precision medicine.

The design of membrane proteins for metabolite loading requires comprehensive consideration of multiple key factors, including balancing hydrophobicity and membrane stability, maintaining metabolite binding activity while ensuring correct folding, and introducing environmentally responsive conformational change modules. Synthetic biology strategies have shown great potential in achieving efficient metabolite enrichment. Specific approaches include construction of membrane localised metabolic factories, development of metabolite capture and fixation systems, and establishment of dynamic regulatory networks such as the use of quorum sensing to control the timing of metabolite synthesis.

In targeted modification, various innovative strategies have been successfully applied to the functional customization of engineered OMVs. Wang et al. developed an OMV-cancer cell membrane hybrid carrier coated on hollow polydopamine (HPDA), which was also used for tumor imaging^[103]. Rezaei et al.^[104] and Sepahdar et al.^[105] introduced OMVs expressing ClyA-EGFR scFv, which demonstrated high affinity for EGFR-positive cancer cells both in vitro and in vivo. Additionally, OMVs overexpressing ClyA-Hy can target hypoxic tumors and remodel the tumor matrix^[106,107].

Besides ClyA fusion proteins, modifying OMVs with glycosylphosphatidylinositol (GPI) anchored proteins is another promising approach. Marianne et al. reported that the lipid portion of GPI anchors inserts into the cell membrane, and two completely different GPI proteins can be displayed on the same surface of OMVs^[108,109]. These multidimensional engineering strategies enhance both metabolite loading efficiency and targeted delivery capabilities of OMVs. They also provide new technological platforms for tumor imaging, treatment, and microenvironment regulation. These advancements demonstrate the broad application potential of synthetic biology in precision medicine, paving new paths for future biomedical research and clinical applications.

In the field of intelligent drug delivery system development, the deep integration of synthetic biology and membrane vesicle engineering is promoting the innovation of drug delivery technology in the direction of precision and controllability. Researchers have built a synthetic OMV cargo platform that is completely decoupled from the host biochemical network, and its drug delivery module can be fully manually designed.

Chen and colleagues designed an OMV based multi module system that uses charge reversal polymers to separate functions, targeting both malignant and immune cells^[110]. In another approach, EVs spontaneously hybridise with nonlamellar liquid crystalline lipid nanoparticles (LCNPs) to form hybrid extracellular vesicles (HEVs). This allows efficient loading of siRNA, mRNA, and nucleic acid aptamers under mild conditions^[110]. In addition to strategies that exploit the physicochemical or biological properties of specific tissues or cells, targeted delivery can also be achieved with the help of external forces, such as the system formed by doxorubicin loaded into placental mesenchymal stem cell derived exosomes and modified with carboxylated Fe₃O₄ nanoparticles, creating an Exo-Dox NP system^[111]. In terms of cargo efficiency improvement technology, a breakthrough has been made in the development of the “Shock Wave Extracellular Vesicle Engineering Technology” (SWEET), which uses shock waves (SWs) to achieve efficient encapsulation of siRNAs^[112]. The bacterial biomineralization system uses a mineral crystal deposition strategy to achieve drug-loading and bactericidal synergy while scavenging Staphylococcus aureus^[113].

These innovative technologies rely on the multidimensional collaboration of modular biological components, including biosensors, molecular switches and orthogonal synthesis systems. They also integrate physical cargo delivery strategies such as electroporation and microfluidics. Together, they redefine OMVs as intelligent nanorobots and lay the technical foundation for the fully artificial construction of synthetic MVs. This enables programmable control from gene circuit design to cargo vesicle assembly, promoting the integration of precision medicine towards molecular level diagnosis and treatment.

In large scale OMV production, the collaborative innovation of strain metabolic engineering and membrane biophysical regulation is driving the simultaneous increase in yield and quality. Researchers have reconstructed bacterial vesicle generation through multidimensional strategies. A team from the University of Waterloo and the Southern University of Science and Technology introduced the eukaryotic membrane curvature regulator EutS into E. coli. This protein induces local outer membrane bending, increasing OMV yield 149-fold while reducing vesicle heterogeneity through optimised membrane tension balance^[76]. At the lipid metabolism level, Wang et al. performed precise gene editing on Yersinia pestis KIM6⁺. They knocked out the lipid A acyltransferase gene lpxP and inserted the phosphatase gene lpxE. This increased OMV yield 2.3-fold and improved biosafety by removing the toxic phosphate group from lipid A^[77].

These advances reveal three core mechanisms for yield enhancement. First, remodeling membrane structural dynamics using curvature regulating proteins such as EutS and the MVSP family. Second, optimising outer membrane stability and vesicle budding efficiency by targeting the lipopolysaccharide synthesis pathway, for example through mutations in waaP or lpxL. Third, lowering the vesicle release energy barrier by adjusting cell wall mechanical strength, such as reducing peptidoglycan crosslinking via dacB knockout. When combined with immunogenicity optimisation strategies, including surface display of meningococcal NadA antigen or chimeric TLR4/MD2 agonist peptides, engineered OMVs can achieve the triple goals of high yield, low toxicity and potent immune activation.

In the future, machine learning assisted design of membrane protein allosteric elements, intelligently coupled with quorum sensing systems such as LuxR and LuxI, may pioneer a third generation OMV biomanufacturing paradigm. This would enable on demand production and directed release.

In large scale production of engineered EVs, the integration of culture condition optimization and advanced bioreactor technology is driving the innovation of production processes towards precision and controllability. By systematically regulating bioreactor parameters, such as increasing the dissolved oxygen content from 30% to 150% in the Neisseria meningitidis culture system, the bacterial oxidative stress pathway can be activated, leading to a fourfold increase in OMVs production^[114]. This metabolic stress based strategy can be combined with optimisation of the carbon to nitrogen ratio, for example by reducing the C/N ratio to induce membrane remodeling, and with regulation of quorum sensing molecule gradients such as 3OC₁₂-HSL. Together these form a multidimensional yield enhancement scheme.

Furthermore, the introduction of optogenetic regulation systems has created a new dimension of spatiotemporal precision control. Cui et al. ^[70]developed conversion microgels (UCMs) that activate programmed colonisation of engineered light responsive bacteria (Lresb) in the intestine through a near infrared to blue light conversion system. The cell adhesion agents secreted by these bacteria increase intestinal colonisation efficiency of EcN 3.2 fold, significantly alleviating DSS induced colitis in mice. This technology platform has been further extended to the field of tumor treatment, achieving a triple synergistic effect of photothermal ablation, gene silencing andimmune activation by simultaneously secreting immune stimulators (such as IL-12) and RNA-loaded OMVs through light controlled engineered bacteria^[115].

These innovative strategies break the limitations of traditional static culture. They construct an intelligent biomanufacturing system with awareness, response, and output capabilities through dynamic regulation of dissolved oxygen, light signals, and quorum sensing molecules. This provides a programmable solution for industrial production and precision medicine applications of EVs. In the future, combining machine learning algorithms to optimise bioreactor parameters in real time will shift EV manufacturing from experience driven to data driven paradigms.

In the field of regenerative medicine, MEVs are also currently used for bone regeneration, demonstrating considerable therapeutic potential. MEVs can influence cellular processes^[145] and can be engineered to express specific targeting molecules on their surfaces through bioengineering strategies. This enables precise cell recognition and integration, thereby improving therapeutic effects and reducing side effects^[146]. Additionally, these MEVs have high loading capacity and can carry growth factors, signaling molecules, or genetic material to stimulate osteoblast proliferation, differentiation and mineralization^[147].

Recombinant probiotics with bone-targeting and osteo inductive abilities were constructed, named MEVs-hCXCR4 (MEVs-C). SOST siRNA was integrated into these MEVs by electroporation to form MEVs-hCXCR4-SOST siRNA (MEVs-CSs), which effectively triggered the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by regulating the WNT signaling pathway^[147]. A novel engineered vesicle named Bone-Targeting Lactobacillus rhamnosus GG Extracellular Vesicle (BT-LGG-EV), utilizing bone-targeting peptides and EVs derived from probiotic LGG was developed. In vitro experiments showed that BT-LGG-EV could increase the formation of mineralized nodules and significantly enhance the expression of osteogenic genes OCN, OSX, OPN, and Runx2. TRAP staining and qPCR analysis indicated that BT-LGG-EV could inhibit osteoclast activity. In an ovariectomized (OVX) mouse model, BT-LGG-EV exhibited strong bone-targeting ability, promoting bone formation and improving osteoporosis by delivering miRNA^[148].

Jansen et al. evaluated the strengths and weaknesses of PCR, NGS, cultivation and FISH. They found that the human gut microbiome is a complex ecosystem composed of diverse microorganisms that play a crucial role in maintaining human health. Furthermore, disruptions in its composition are closely linked to a variety of health problems^[149]. Another study discovered that colonization with the child gut microbiota (CGM), rather than the elderly gut microbiota, could prevent bone loss and the reduction of bone strength in ovariectomized osteoporosis mice. EVs derived from the CGM protected against osteoporosis by targeting Akkermansia muciniphila (Akk), enhancing osteogenic activity and inhibiting osteoclast formation^[150]. Furthermore, Wang et al. demonstrated that OMVs of Gram-negative bacterium Providencia (P.M.) have the ability to inhibit osteoclast formation and bone resorption. These OMVs alleviated bone loss in experimental osteoporosis and rheumatoid arthritis by downregulating miR-96-5p^[151]. These findings provide new approaches for developing bone regeneration strategies using bacteria.

In summary, MEVs show significant therapeutic potential in the field of bone regeneration. They effectively promote osteoblast proliferation and differentiation through precise cell recognition and loading of bioactive molecules. However, challenges remain in scale-up production, standardization, and clinical translation.

In the field of dermatopathology, MEVs have shown great promise in regulating skin immune responses, promoting wound healing, and preventing scar formation. The applicability and effectiveness of Lactobacillus druckerii-derived extracellular vesicles (LDEVs) in treating hypertrophic scars (HS) were reported by Han et al.^[152]. In vitro experiments and animal models showed that LDEVs significantly inhibited the expression of collagen and α-smooth muscle actin (α-SMA) in fibroblasts, reduced cell proliferation, and promoted skin cell proliferation, neovascularization, and wound healing, thereby reducing the formation of hypertrophic scars.

Extracellular vesicles (SEStaphylococcus epidermidis mitigate inflammatory responses in a mouse model of atopic dermatitis (AD) by reducing proinflammatory gene expression, enhancing skin barrier function, and promoting keratinocyte proliferation and migration^[153]. Additionally, SE-EVs increase the expression of human β-defensin and enhance resistance to Staphylococcus aureus by activating the Toll-like receptor 2 (TLR2) pathway. In the AD mouse model, application of SE-EVs was observed to significantly reduce inflammatory cell infiltration, TH2 cytokine gene expression, and IgE levels, indicating that SE-EVs may serve as effective bioactive nanocarriers for treating this condition. These findings provide a scientific basis for using EVs from commensal skin microbiota as a novel therapeutic strategy.

Chen et al. revealed that membrane vesicles (RMVs) secreted by Lactobacillus reuteri induce macrophages ploraization towards an anti-inflammatory state, enhancing mucosal and skin wound healing^[154]. The study showed that RMVs could reduce the number of proinflammatory macrophages in inflamed tissues and regulate mitochondrial permeability of macrophages through their internal 3-hydroxypropionaldehyde (3-HPA) component. This led to reduced oxidative stress, prompting macrophage phenotypic transformation toward an anti-inflammatory state, which is conducive to wound healing. The potential of RMVs to promote wound healing was confirmed in both in vitro and in vivo experiments, providing a new method for using probiotic EVs to treat skin injuries.

Collectively, these studies indicate that MEVs hold great potential as innovative and clinically applicable therapies for skin diseases. By regulating host immune responses and promoting wound healing, MEVs offer new treatment approaches in dermatopathology and are expected to play a key role in future therapeutic strategies.

MEVs have attracted extensive attention due to their potential therapeutic applications in various diseases. Preclinical studies have shown their abilities to promote placental development, alleviate preeclampsia, regulate biofilm formation, reduce stress-induced changes in the brain, and improve depressive behavior^[144,155]. Notably, MEVs derived from probiotics were found to induce the expression of neurotrophic factors in the hippocampus, thereby countering stress-induced neuronal dysfunction^[156]. Additionally, MEVs derived from Lactococcus lactis have been shown to stimulate IL-12 production, activate dendritic cells, and alter immune responses in allergic asthma, thereby reducing airway inflammation^[157]. However, the clinical translation of MEVs requires in-depth understanding of their safety and the establishment of standardized regulatory frameworks. Further research is crucial for optimizing BEV production, purification, and characterization, with a focus on improving their delivery efficiency to targets.

Exploring various delivery systems, including hydrogels, scaffolds, and nanoparticles, may enhance the stability, targeting, and controlled release of MEVs. Ultimately, long term safety and efficacy studies are essential for establishing the role of MEVs in regenerative medicine treatments. Wu et al. proposed a method for supramolecular covalent cascade modification of cell membranes, through which biomimetic hydrogel materials (SFSHs) that mimic skin structure and function can be prepared^[158]. This method uses bacterial outer membrane vesicles as crosslinking surfaces. Compared with traditional hydrogels formed by small molecule crosslinkers, SFSHs have several advantages. The deformation of vesicles can dissipate energy, thereby greatly improving the mechanical strength of SFSHs. Additionally, MEVs carry many bioactive substances derived from bacteria, which have unique inhibitory effects on pathogenic bacteria and can promote dendritic cell maturation. This study provides ideas for developing biomimetic skin materials with tunable structural and functional properties and exemplifies the feasibility of integrating MEVs with materials, including hydrogels for tissue engineering applications.

In summary, selecting biomaterials for loading MEVs is crucial. In recent studies, polyhydroxyalkanoates (PHA) have been widely used in tissue regeneration research due to their favorable biocompatibility and biodegradability. Similarly, it was reported that PHA can be used to load and deliver MEVs, which is a promising approach for developing advanced therapeutic strategies^[159,160].