Repurposing rabeprazole sodium as an anti-Clostridium perfringens drug by inhibiting perfringolysin O.

AIMS: Clostridium perfringens infections affect food safety, human health, and the development of the poultry feed industry. Anti-virulence is an alternative strategy to develop new drug. Perfringolysin O (PFO) is an exotoxin of C. perfringens that has been demonstrated to play critical roles in the pathogenesis of this organism, promising it an attractive target to explore drugs to combat C. perfringens infection. METHODS AND RESULTS: Based on an activity-based screening, we identified six PFO inhibitors from the Food and Drug Administration (FDA)-approved drug library, among which rabeprazole sodium (RS) showed an optimal inhibitory effect with an IC50 of 1.82 ± 0.746 µg ml-1. The GLY57, ASP58, SER190, SER193-194, ASN199, GLU204, ASN377, THR379, and ALA200 in PFO interacted with RS during binding based on an energy analysis and H-bond analysis. This interaction blocked the oligomer formation of PFO, thereby inhibiting its cytotoxicity. RS treatment significantly increased the survival rate and alleviated pathological damage in C. perfringens or PFO-treated Galleria mellonella. CONCLUSIONS: RS could potentially be used as a candidate drug for treating C. perfringens infection.

Construction of Fusion Protein with Carbohydrate-Binding Module and Leaf-Branch Compost Cutinase to Enhance the Degradation Efficiency of Polyethylene Terephthalate.

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCCICCG shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCCICCG, and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCCICCG-TrCBM and CfCBM-LCCICCG were measured to be 70 and 80 °C, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 °C for 300 min. Compared with LCCICCG, the binding affinity of LCCICCG-TrCBM and CfCBM-LCCICCG for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability.

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus.

Diosgenin is an important raw material used in the synthesis of steroid drugs, and it is widely used in the pharmaceutical industry. The traditional method of producing diosgenin is through using raw materials provided via the plant Dioscorea zingiberensis C. H. Wright (DZW), which is subsequently industrially hydrolyzed using a high quantity of hydrochloric and sulfuric acids at temperatures ranging from 70 °C to 175 °C. This process results in a significant amount of unmanageable wastewater, creates issues of severe environmental pollution and consumes high quantities of energy. As an alternative, the enzymolysis of DZW to produce diosgenin is an environmentally and friendly method with wide-ranging prospects for its application. However, there are still only a few enzymes that are suitable for production on an industrial scale. In this study, three new key enzymes, E1, E2, and E3, with a high conversion stability of diosgenin, were isolated and identified using an enzyme-linked-substrate autography strategy. HPLC-MS/MS identification showed that E1, a 134.45 kDa protein with 1019 amino acids (AAs), is a zinc-dependent protein similar to the M16 family. E2, a 97.89 kDa protein with 910 AAs, is a type of endo-ß-1,3-glucanase. E3, a 51.6 kDa protein with 476 AAs, is a type of Xaa-Pro aminopeptidase. In addition, the method to immobilize these proteins was optimized, and stability was achieved. The results show that the optimal immobilization parameters are 3.5% sodium alginate, 3.45% calcium chloride concentration, 1.4 h fixed time, and pH 8.8; and the recovery rate of enzyme activity can reach 43.98%. A level of 70.3% relative enzyme activity can be obtained after employing six cycles of the optimized technology. Compared with free enzymes, immobilized enzymes have improved stability, acid and alkaline resistance and reusability, which are conducive to large-scale industrial production.

An Atom-Economic Enzymatic Cascade Catalysis for High-Throughput RAFT Synthesis of Ultrahigh Molecular Weight Polymers.

High-throughput synthesis of well-defined, ultrahigh molecular weight (UHMW) polymers by green approaches is highly desirable but remains unexplored. We report the creation of an atom-economic enzymatic cascade catalysis, consisting of formate oxidase (FOx) and horseradish peroxidase (HRP), that enables high-throughput reversible addition-fragmentation chain transfer (RAFT) synthesis of UHMW polymers at volumes down to 50 µL. FOx transforms formic acid, a C1 substrate, and oxygen to CO2 and H2 O2 , respectively. CO2 can escape from solution while H2 O2 is harnessed in situ by HRP to generate radicals from acetylacetone for RAFT polymerization, leaving no waste accumulation in solution. Oxygen-tolerant RAFT polymerization using enzymatic cascade redox cycles was successfully performed in vials and 96-well plates to produce libraries of well-defined UHMW polymers, and represents the first example of high-throughput synthesis method of such materials at extremely low volumes.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

An ATP-Responsive Codelivery System of Doxorubicin and MiR-34a To Synergistically Inhibit Cell Proliferation and Migration.

Establishing stimulus-responsive nanosystems for the codelivery of anticancer drug and oligonucleotide is a promising strategy in cancer treatment owing to the combination of chemotherapy and gene therapy in a synergistic manner. Herein, an ATP aptamer and its cDNA sequence were first hybridized to produce the duplex, into which chemotherapeutic agent doxorubicin (DOX) interacted through the GC-rich motif of duplex, and PEI25K was then employed as a carrier to condense the DOX-loading duplex and miR-34a to construct the ternary nanocomplex PEI/DOX-Duplex/miR-34a. The nanocomplex exhibited a favorable drug release profile through the response to high concentration of ATP in the cytosol. The ATP-responsive delivery system was demonstrated to possess higher antiproliferative effect (cell viability of <40%) than the single cargo delivery, which could be attributed to the synergistic induction of cell apoptosis and cell cycle arrest from DOX and miR-34a. Furthermore, wound healing and Transwell assay elucidated the higher antimigration effect of ternary nanocomplex than DOX-Duplex or miR-34a delivery. Overall, the combinatorial delivery of DOX and miR-34a through an ATP-responsive manner could trigger the rapid release of cargoes in the cytosol and enhance the inhibition of cell proliferation and migration through the synergistic manner of these two components.

Improving the Intracellular Drug Concentration in Lung Cancer Treatment through the Codelivery of Doxorubicin and miR-519c Mediated by Porous PLGA Microparticle.

Porous PLGA microparticle for the coencapsulation of doxorubicin and miR-519c was successfully constructed through the water-oil-water emulsion solvent evaporation method, using ammonium bicarbonate as a porogen. It has been characterized with high porous surface, adaptive aerodynamic diameter (<10 µm), favorable drug loading, and sustained release profile. The release supernatant exhibited a higher inhibition of cell proliferation than those from porous PLGA microparticles harboring a single component (doxorubicin or miR-519c), attributing to the enhanced induction of cell apoptosis and cell cycle arrest at S phase. Finally, the improved intracellular concentration of doxorubicin was elucidated by flow cytometry and liquid chromatography with tandem mass spectrometry, owing to the knockdown of drug transporter ABCG2 by miR-519c. Overall, the porous PLGA microparticle combining chemotherapy and gene therapy could facilitate the antitumor efficacy and reduce the side effects, and thus, it is potential to be used as a sustained release system for lung cancer treatment via pulmonary administration.

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

Facile synthesis of block copolymers by tandem ROMP and eROP from esters precursors.

In the present study, block copolymers were first synthesized through a tandem ring-opening metathesis polymerization (ROMP) and conventional enzymatic ring-opening polymerization (eROP) from hydroxyl initiator. Furthermore, a novel synthesis route, single-step eROP from ester precursor was successfully developed to synthesize targeted copolymers. The as-prepared polymers were analyzed by NMR, GPC, DSC, and MALDI-TOF-MS. There was no difference in the characteristic peaks of NMR between the end products obtained from these two synthetic routes. The GPC data showed that the copolymer obtained from single-step eROP was similar to the end product obtained from the traditional multistep synthesis method. Afterward, we used model compounds to carry out the conventional eROP and the single-step eROP. Finally, through the kinetic analysis and structural analysis of the resulting product, a reasonable initiation mechanism for this single-step eROP was elucidated.

Glutaraldehyde cross-linking of immobilized thermophilic esterase on hydrophobic macroporous resin for application in poly(ε-caprolactone) synthesis.

The immobilized thermophilic esterase from Archaeoglobus fulgidus was successfully constructed through the glutaraldehyde-mediated covalent coupling after its physical adsorption on a hydrophobic macroporous resin, Sepabeads EC-OD. Through 0.05% glutaraldehyde treatment, the prevention of enzyme leaching and the maintenance of catalytic activity could be simultaneously realized. Using the enzymatic ring-opening polymerization of ε-caprolactone as a model, effects of organic solvents and reaction temperature on the monomer conversion and product molecular weight were systematically investigated. After the optimization of reaction conditions, products were obtained with 100% monomer conversion and Mn values lower than 1010 g/mol. Furthermore, the cross­linked immobilized thermophilic esterase exhibited an excellent operational stability, with monomer conversion values exceeding 90% over the course of 12 batch reactions, still more than 80% after 16 batch reactions.

(De)carboxylation mechanisms of heteroaromatic substrates catalyzed by prenylated FMN-dependent UbiD decarboxylases: An in-silico study.

The UbiD enzymes are proposed to catalyze reversible (de)carboxylation reaction of unsaturated carboxylic acids using prenylated flavin mononucleotide (prFMN) as a cofactor. This positions UbiD enzymes as promising candidates for converting CO2 into valuable chemicals. However, their industrial-scale biotransformation is currently constrained by low conversion rates attributed to thermodynamic limitations. To enhance the carboxylation activity of UbiD enzymes, a molecular-level understanding of the (de)carboxylation mechanisms is necessary. In this study, we investigated the reaction mechanisms of heteroaromatic substrates catalyzed by PtHmfF, PaHudA, and AnlnD enzymes using molecular dynamics (MD) simulations and free energy calculations. Our extensive mechanistic study elucidates the mechanisms involved in the formation of the initial prFMN-substrate intermediate. Specifically, we observed nucleophilic attack during decarboxylation, while carboxylation reactions involving furoic acid, pyrrole, and indole tend to favor a 1,3-dipolar cycloaddition mechanism. Furthermore, we identified proton transfer as the rate-limiting step in the carboxylation reaction. In addition, we considered the perspectives of reaction energies and electron transfer to understand the distinct mechanisms underlying decarboxylation and carboxylation. Our calculated free energies are consistent with available experimental kinetics data. Finally, we explored how different rotamers of catalytic residues influence the efficiency of the initial intermediate formation.

Enzymes encapsulated in organic-inorganic hybrid nanoflower with spatial localization for sensitive and colorimetric detection of formate.

Formate is an important environmental pollutant, and meanwhile its concentration change is associated with a variety of diseases. Thus, rapid and sensitive detection of formate is critical for the biochemical analysis of complex samples and clinical diagnosis of multiple diseases. Herein, a colorimetric biosensor was constructed based on the cascade catalysis of formate oxidase (FOx) and horseradish peroxidase (HRP). These two enzymes were co-immobilized in Cu3(PO4)2-based hybrid nanoflower with spatial localization, in which FOx and HRP were located in the shell and core of nanoflower, respectively (FOx@HRP). In this system, FOx could catalyze the oxidation of formate to generate H2O2, which was then utilized by HRP to oxidize 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid to yield blue product. Ideal linear correlation could be obtained between the absorbance at 420 nm and formate concentration. Meanwhile, FOx@HRP exhibited excellent detection performance with low limit of detection (6 µM), wide linear detection range (10-900 µM), and favorable specificity, stability and reusability. Moreover, it could be applied in the detection of formate in environmental, food and biological samples with high accuracy. Collectively, FOx@HRP provides a useful strategy for the simple and sensitive detection of formate and is potentially to be used in biochemical analysis and clinical diagnosis.

Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

A quaternary ammonium-based nanosystem enables delivery of CRISPR/Cas9 for cancer therapy.

Genome editing mediated by CRISPR/Cas9 is an attractive weapon for cancer therapy. However, in vivo delivery of CRISPR/Cas9 components to achieve therapeutic efficiency is still challenging. Herein, a quaternary ammonium-functionalized poly(L-lysine) and a cholesterol-modified PEG (QNP) were self-assembled with a negatively charged CRISPR Cas9/sgRNA ribonucleoprotein (RNP) to form a ternary complex (QNP/RNP). Such a delivery system of QNP exhibited multiplex genome editing capabilities in vitro (e.g., the GFP gene and the PLK1 gene). In addition, QNP/RNPPLK1 containing PLK1 sgRNA led to 30.99% of genome editing efficiency in MCF-7 cells with negligible cytotoxicity of the carrier. QNP/RNPPLK1, which was capable of simultaneously inhibiting cell proliferation, mediating cell cycle arrest and downregulating expression of PLK1, held great in vitro therapeutic efficiency. Moreover, QNP/RNPPLK1 exhibited outstanding accumulation in tumors and high biocompatibility in vivo. In an MCF-7 xenograft animal model, QNP/RNPPLK1 showed excellent anti-tumor efficacy and achieved 17.75% indels ratio. This work showcases the successful delivery of CRISPR Cas9/sgRNA RNP with enhanced genome editing efficiency and provides a potential on-demand strategy for cancer therapy.

Mechanistic insights into the conversion of flavin adenine dinucleotide (FAD) to 8-formyl FAD in formate oxidase: a combined experimental and in-silico study.

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH-. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH- group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties.

Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo.

IMPORTANCE: Salmonella spp. remains a major worldwide health concern that causes significant morbidity and mortality in both humans and animals. The spread of antimicrobial resistant strains has declined the efficacy of conventional chemotherapy. Thus, novel anti-infection drugs or strategies are needed. Anti-virulence strategy represents one of the promising means for the treatment of bacterial infections. In this study, we found that the natural compound fisetin could inhibit Salmonella invasion of host cells by targeting SPI-1 regulation. Fisetin treatment impaired the interaction of the regulatory protein HilD with the promoters of its target genes, thereby suppressing the expression of T3SS-1 effectors as well as structural proteins. Moreover, fisetin treatment could reduce pathology in the Salmonella murine infection model. Collectively, our results suggest that fisetin may serve as a promising lead compound for the development of anti-Salmonella drugs.

Immobilization of Bacillus thuringiensis Cry1Ac in metal-organic frameworks through biomimetic mineralization for sustainable pest management.

Traditional chemical pesticide dosage forms and crude application methods have resulted in low pesticide utilization, increased environmental pollution, and the development of resistance. Compared to traditional pesticides, nanopesticides enhance the efficiency of pesticide utilization and reduce the quantity required, thereby decreasing environmental pollution. Herein, Cry1Ac insecticidal crystal protein from Bacillus thuringiensis Subsp. Kurstaki HD-73 was encapsulated in a metal-organic framework (zeolite imidazolate framework-8, ZIF-8) through biomimetic mineralization to obtain Cry1Ac@ZIF-8 nanopesticides. The Cry1Ac@ZIF-8 nanopesticides exhibited a dodecahedral porous structure, and the introduction of Cry1Ac did not affect the intrinsic crystal structure of ZIF-8. The indoor toxicity analysis revealed that the toxicity of Cry1Ac towards Ostrinia furnacalis (Guenée), Helicoverpa armigera Hubner, and Spodoptera litura Fabricius was not affected by ZIF-8 encapsulation. Surprisingly, Cry1Ac@ZIF-8 still exhibited excellent pest management efficacy even after exposure to heat, UV irradiation, and long-term storage. More importantly, the encapsulation of ZIF-8 significantly enhanced the internal absorption performance of Cry1Ac in maize leaves and extended its persistence period. Thus, ZIF-8 could potentially serve as a promising carrier for the preparation of nanopesticides with enhanced applicability, stability, and persistence period, providing a powerful strategy to improve the application of Cry1Ac in future agricultural pest management.

Silibinin triggers apoptosis and cell-cycle arrest of SGC7901 cells.

Silibinin, a flavonoid compound, has shown to be of chemopreventive potential against many cancers. However, its efficacy against gastric cancer has not been well elucidated. Here, we assessed the activity of Silibinin on apoptosis and cell-cycle arrest in human gastric cells culture system using SGC-7901 as the model. Silibinin treatment could inhibit the cell growth and cause a prominent G2 phase arrest and apoptosis in dose- and time-dependent manner. In mechanistic studies, Silibinin decreased the protein level of p34cdc2, which might be the possible molecular mechanism of Silibinin efficacy on the growth inhibition in SGC-7901 cells. In addition, Silibinin caused an increase in p53 and p21 protein level as well as mRNA levels. Interestingly, Silibinin-induced apoptosis in SGC-7901 cells was independent of caspases activation. These results indicated that Silibinin is a cell-cycle regulator and apoptosis inducer in human gastric carcinoma SGC-7901 cells and might be used as a candidate chemopreventive agent for gastric carcinoma prevention and intervention.

Fluorinated polyamidoamine dendrimer-mediated miR-23b delivery for the treatment of experimental rheumatoid arthritis in rats.

In rheumatoid arthritis (RA), insufficient apoptosis of macrophages and excessive generation of pro-inflammatory cytokines are intimately connected, accelerating the development of disease. Here, a fluorinated polyamidoamine dendrimer (FP) is used to deliver miR-23b to reduce inflammation by triggering the apoptosis of as well as inhibiting the inflammatory response in macrophages. Following the intravenous injection of FP/miR-23b nanoparticles in experimental RA models, the nanoparticles show therapeutic efficacy with inhibition of inflammatory response, reduced bone and cartilage erosion, suppression of synoviocyte infiltration and the recovery of mobility. Moreover, the nanoparticles accumulate in the inflamed joint and are non-specifically captured by synoviocytes, leading to the restoration of miR-23b expression in the synovium. The miR-23b nanoparticles target Tab2, Tab3 and Ikka to regulate the activation of NF-κB pathway in the hyperplastic synovium, thereby promoting anti-inflammatory and anti-proliferative responses. Additionally, the intravenous administration of FP/miR-23b nanoparticles do not induce obvious systemic toxicity. Overall, our work demonstrates that the combination of apoptosis induction and inflammatory inhibition could be a promising approach in the treatment of RA and possibly other autoimmune diseases.

Exosome-mediated delivery of superoxide dismutase for anti-aging studies in Caenorhabditis elegans.

Aging is a dynamic and progressive process mediated by reactive oxygen species (ROS), and the antioxidant enzyme superoxide dismutase (SOD) can effectively scavenge ROS to extend longevity. However, the instability and impermeability of native enzyme limit its in vivo biomedical application. Currently, exosome as protein carriers attracts considerable attention in the disease treatment owing to low immunogenicity and high stability. Herein, SOD was encapsulated into exosomes via mechanical extrusion with saponin permeabilization to obtain SOD-loaded EXO (SOD@EXO). SOD@EXO with a hydrodynamic diameter of 101.7 ± 5.6 nm could scavenge excessive ROS and protect the cells from oxidative damage induced by 1-methyl-4-phenylpyridine. Compared with native SOD, SOD@EXO significantly extended the lifespan of N2 wild-type Caenorhabditis elegans under normal conditions. Moreover, SOD@EXO improved the resistance against heat and oxidative stress, leading to notable survival ratio under these hostile conditions. Overall, the exosome-mediated delivery of SOD could reduce ROS level and delay aging in C. elegans model, thereby providing potential strategies to treat ROS-related diseases in future.