Extracellular vesicle engineering using a small scaffold protein

Domain: biology
Published: 2026-03-10
Editorial outcome: accept

Abstract
Extracellular vesicles (EVs) are promising drug-delivery vehicles owing to their biocompatibility and low immunogenicity. Genetic engineering of a membrane-bound EV-sorting scaffold protein empowers EVs by installing targeting moieties on the surface and enriching therapeutic cargo in the lumen. However, the choice of scaffold proteins with simple structures and short sequences is limited. Here, we conduct mass spectrometry-based proteomic studies and identify ENPP1 as a superior scaffold protein. Furthermore, we show that a truncated 144-amino acid variant, EN144, efficiently loads diverse therapeutic cargoes and outperforms conventional scaffolds. By fusing EN144 to the IL-6 decoy receptor gp130, we create engineered decoy EVs that potently inhibit inflammatory IL-6 trans-signaling. In mouse models, these EVs reduce inflammation, improve survival in sepsis, and, when targeted to cartilage, alleviate tissue damage in osteoarthritis. Our work establishes EN144 as a minimal, high-performance scaffold for EV engineering and demonstrates its broad therapeutic potential for inflammatory diseases.

Introduction
Extracellular vesicles (EVs) have emerged as a promising avenue for targeted drug delivery. These nanoscale particles, naturally secreted by cells, facilitate vital intercellular transport processes1. Compared to other delivery modalities like nanoparticles and liposomes, EVs possess high biocompatibility, extended circulation half-lives, minimal toxicity, and the ability to traverse biological barriers2. EVs can encapsulate biomolecules via exogenous or endogenous loading. Exogenous loading, applicable to pre-isolated EVs, involves physical treatment (sonication, electroporation, chemical conjugation) and is suitable for small payloads like microRNAs and low-molecular-weight compounds. These procedures, however, may incur RNA precipitation and EV damage3. Alternatively, endogenous loading leverages the overexpression of biomolecules fused to EV-sorting proteins naturally expressed in EVs during their biogenesis within cells. This approach preserves the membrane integrity of EVs and enables the loading of large cargoes such as proteins. However, the loading efficacy relies on the abundance of the sorting protein4, 5, 6. Also, some EV-sorting proteins may be expressed only in a subpopulation of the purified EVs, affecting the final product's homogeneity7. Therefore, EV-sorting proteins with a uniformly high expression level and robust engineerability as the scaffold protein are highly desired. Recent research employing fluorescent enzyme-based assays has uncovered that an EV-sorting protein TSPAN2 surpasses CD63, exhibiting a higher signal level of luciferase reporter ThermoLuc within EVs (93% vs. 89%)5. Notably, these proteins belong to the multifunctional tetraspanin superfamily of multi-pass transmembrane proteins. As tetraspanins are known to be involved in the initiation, promotion, and progression of tumorigenesis8, their use as scaffold proteins raises concerns. Significant endeavors have been made to identify alternative candidates, which resulted in the discovery of PTGFRN, BASP1, and TSPAN14 as highly potential scaffold proteins6, 9. However, most of these studies employ a single method for EV extraction when screening for scaffold proteins, overlooking the inherent risk of potential protein contamination associated with any particular extraction technique. Therefore, a holistic approach to identifying scaffold proteins, ideally with minimalist structure and sequence, homogenous expression, and efficient cargo loading, is highly desirable.

On another note, inflammation is a complex medical condition that orchestrates diverse physiological and pathological responses to stimuli, primarily geared toward tissue repair and pathogen clearance10. Chronic inflammation may develop when the delicate balance is interrupted, and diseases such as sepsis and osteoarthritis (OA) may be triggered. Sepsis arises from a malfunction in the body’s systemic inflammatory and immune response to infection, resulting in organ dysfunction11. The exaggerated inflammatory reaction, characterized by cytokine storms, has been long recognized as the primary driver of sepsis-related mortality12. OA, a degenerative disorder, is marked by the progressive loss of articular cartilage accompanied by chronic inflammation13. A crucial aspect in the development of OA is the disruption of cytokine homeostasis, where pro-inflammatory cytokines play a pivotal role. By activating catabolic enzymes like matrix metalloproteinase 13 (MMP13), these cytokines contribute to the degradation of cartilage and other vital intra-articular structures14. Aberrant or heightened production of cytokines, including interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and IL-1β, potentially lead to organ failure and even death15. Currently, anti-cytokine treatment has emerged as a promising therapeutic approach for inflammatory diseases16. For example, monoclonal antibodies targeting inflammatory cytokines, such as Tocilizumab, have been approved as first-line therapies for treating rheumatoid arthritis (RA).

Among all the cytokines, interleukin 6 (IL-6) is essential in the inflammatory cascade of sepsis and OA, thus raising the speculation of targeting the IL-6 signaling pathway for anti-inflammatory therapy against these diseases17, 18, 19. In the classic signaling mechanism, IL-6 binds to the membrane-bound IL-6 receptor (mIL-6R), forms a complex that engages with IL6ST (gp130), and triggers intracellular signaling cascades20. Conversely, in trans-signaling, IL-6 combines with soluble IL-6 receptor (sIL-6R) to form an IL-6:sIL-6R complex, which subsequently interacts with gp130, activating the JAK signaling pathway20. Recent studies have revealed IL-6 as a dual-nature cytokine exhibiting both anti-inflammatory and pro-inflammatory effects through classic signaling and trans-signaling pathways, respectively10. However, antibodies targeting IL-6 or IL-6R, such as tocilizumab, cannot differentiate between these two pathways21. Thus, therapeutic approaches that preserve the anti-inflammatory properties of classic signaling while selectively inhibiting the pro-inflammatory trans-signaling of IL-6 are highly beneficial but challenging. One such strategy involves leveraging soluble gp130 (sgp130) shed from the plasma membrane. Sgp130 is a natural inhibitor of IL-6 trans-signaling by sequestering the IL-6:sIL-6R complex22. Recent studies report that TNFR1-enriched EVs function as a TNF-α decoy, inhibiting TNF-α-induced p38 MAPK phosphorylation23. Decoy EVs with neutralizing functions are emerging therapeutic tools24, 25, 26. We then envision that EVs displaying gp130 may inhibit IL-6 trans-signaling and present an anti-inflammatory therapy for sepsis and OA treatment.

Here, we screen proteins expressed on the surface of EVs separated from Expi239F cells (Expi239F-EVs) using three distinct EV extraction strategies and proteomics analysis and identify an effective EV-sorting protein ENPP1. Notably, we identify a truncated version of ENPP1 (EN144), with only 144 amino acids, as one of the shortest and structurally simplest scaffold proteins. EVs carrying overexpressed EN144 (EN144-EVs) show improved loading of biomolecules in the lumen, high safety and stability, and preferential in vivo distribution to the liver and spleen in animals. EN144-EVs displaying gp130 in the lumen and the targeting motif on the surface simultaneously through engineered EN144 demonstrate a potent anti-inflammatory effect in animal models of sepsis and OA.

Results
Identification of EV-sorting proteins in Expi293F-EVs
Because of their robust growth in chemically defined serum-free media, Expi293F cells (derivatives of the 293 cell line) are an ideal chassis for large-scale, controlled production of biologics that meet the quality requirements of clinical applications27. We purified EVs from Expi293F cells, designated as Expi293F-EVs, by following the established protocol28. Expi293F-EVs exhibited cup-shaped double-membrane structures, typical of mammalian-cell-derived EVs, with a size distribution of 67 ± 30 nm (Supplementary Figures 1a, b). EV-specific marker proteins, such as Hsp70, TSG101, and CD9, but not the endoplasmic reticulum marker Calnexin or the core component of nucleosome (Histone H3), were found in the Expi293F-EVs preparation (Supplementary Figure 1c), suggesting the absence of nuclear fragments and ER-derived contaminants. We employed three different and independent strategies to isolate EVs: ultracentrifugation, magnetic bead-based EVtrap technology29, and a purification process integrating tangential flow industrial extraction30 with size exclusion chromatography. Next, we conducted comprehensive proteomic analyses on samples from the three isolation methods. Different sources of EVs were used to mitigate any possible bias or false hits of each isolation method (Figure 1a). A panel of proteins was selected as candidates for EV-sorting proteins and divided into four categories: (1) abundant proteins in Expi293F-EVs, (2) membrane proteins, and (3) proteins that have not been reported as EV-sorting proteins5, 6, 9, 31, and (4) proteins involved in EV biogenesis and release, including Rab7a, VTA1, Syntenin-1, and IST1. A total of 15 candidate proteins were identified, including ENPP1, Rab7a, STX7, CXADR, EPCAM, TFRC, VTA1, Syntenin-1, SNAP23, AT1A1, STX4, PDL1, VAMP2, CD55, and IST1 (Supplementary Table 4). Notably, among the list, Rab7a is not a membrane-bound protein.

Next, we assess the EV-sorting capability of the 15 candidate proteins by fusing them with the green fluorescent protein EGFP (Supplementary Table 4). Briefly, EGFP was appended to either the N- or C-terminus of the candidates, stable Expi293F cell lines were established using plasmids encoding EGFP fusion, and EVs carrying the fluorescent candidate proteins were isolated and characterized (Figure 1b, Supplementary Figure 2a). All the engineered EVs showed particle sizes of 50-150 nm, suggesting that overexpressing the candidate proteins did not significantly affect the EV packaging (Supplementary Figure 2b). Based on western blot (WB) analysis, EVs from Expi293F cells overexpressing ENPP1, Rab7a, STX7, CXADR, SNAP23, PDL1, EPCAM, and VTA1 exhibited elevated EGFP expression levels compared to other groups, surpassing that of EVs derived from Expi293F cells overexpressing PTGFRN (named PTGFRN-EVs, Figure 1b, Supplementary Figure 3). Among these positive hits, EVs from Expi293F cells overexpressing ENPP1 (ENPP1-EVs) and Rab7a (Rab7a-EVs) demonstrated the highest EGFP levels. Conversely, EVs derived from Expi293F cells overexpressing TFRC, TFRC-8S1 (the transmembrane sequence of TFRC), Syntenin-1, AT1A1, STX4, VAMP2, CD55, IST1, pDisplay, and Lamp2b showed relatively low EGFP signals (Figure 1b, Supplementary Figure 3). Additionally, EVs from Expi293F overexpressing unfused EGFP failed to yield detectable protein bands, indicating that EGFP protein does not spontaneously sort into EVs without fusion to an EV-sorting protein (Figure 1b, Supplementary Figure 3).

Next, seven top-performing candidate proteins alongside two control sorting proteins, PTGFRN and Lamp2b, were selected for further assessment using mass spectrometry. When analyzed from an equal number of EV particles, ENPP1-EVs and Rab7a-EVs exhibited the highest raw EGFP LFQ signals, 5.23-fold and 12.07-fold higher than that of PTGFRN-EVs, respectively (Figure 1c). Semi-quantitative protein analysis indicated that the levels of EGFP protein loaded into ENPP1-EVs and Rab7a-EVs were approximately three- and four-fold higher, respectively, than those in PTGFRN-EVs (Figure 1d, Supplementary Figure 4). Nanoscale flow cytometry confirmed that ENPP1 and Rab7a overexpression led to abundant and uniform EGFP distribution in EVs, with a loading capacity exceeding 90% (Figure 1e). These findings indicate that ENPP1 and Rab7a can serve as potent EV-sorting proteins to facilitate efficient cargo loading.

Subsequently, we assess the entry of ENPP1-EVs, Rab7a-EVs, and PTGFRN-EVs into Expi293F cells. Although Rab7a-EVs exhibited the highest EGFP loading (Figure 1d), they were internalized less efficiently than ENPP1-EVs and PTGFRN-EVs (Supplementary Figure 5a). Therefore, we focused on ENPP1-EVs in the following experiment and explored ENPP1 as a potential scaffold protein for EV engineering (Rab7b, in contrast, is not membrane-bound, so it is an EV-sorting protein, but not a scaffold protein). WB analysis revealed a time-dependent increase in the entry of ENPP1-EVs into recipient cells, which saturated at 18 h (Supplementary Figure 5b, c). As smaller and simpler EV-sorting scaffold proteins (<50 kDa) facilitate cargo engineering9, we next aim to test ENPP1 truncations by cutting at the domain boundaries (Figure 1f). Transfection efficiencies of truncated and full-length ENPP1 plasmids ranged from 38% to 58% (Supplementary Figure 6a). EVs expressing the 144-amino-acid truncation, EN144 (EN144-EVs), exhibited a marked enhancement in cargo loading, a three-fold increase over ENPP1-EVs (Figure 1g, Supplementary Figure 6b). In addition to the Expi293F cell line, other adherent cell lines are frequently employed as sources of EVs. Based on the ratio of EN144 in EVs to all the EN144 in the cell lysates (the total expression levels varied in different cells), we found that a similar percentage of EN144 was enriched in EVs when we overexpressed EN144 in cell lines, including 293T cells, SF-MSCs, HeLa cells, and HcerEpic cells. These results demonstrated that EN144 exhibited comparable sorting efficiency in various cell types (Supplementary Figure 7a, b). In contrast to PTGFRN, Lamp2b, and ENPP1, EN144 has only 144 amino acids with a significantly simpler transmembrane architecture (Figure 1h). Taken together, based on multiple screening strategies, we identified a truncated version of ENPP1, comprising only 144 amino acids, as one of the most efficient EV-sorting proteins and, to our knowledge, the smallest and simplest EV-specific scaffold protein.

Biocompatibility and bioavailability test
Next, we characterized the physical properties, toxicity, and in vivo distribution of EN144-EVs. WB results indicated EN144 was highly expressed in the lysates of the EN144-transfected Expi293F and EN144-EVs (Figure 2a). TEM imaging confirmed their spherical morphology and lipid bilayer structure (Figure 2b). Resistive pulse sensing (RPS) analysis revealed that EN144-EVs have similar sizes as Expi293F-EVs (72 nm vs. 66 nm, Figure 2c). Both EN144-EVs and Expi293F-EVs expressed characteristic biomarkers Hsp70, TSG101, and Syntenin-1 but not Calnexin or Histone H3 (Figure 2d). LC-MS/MS-based proteomics was performed to assess the impact of EN144 on EV protein profiles, revealing 143 upregulated and 45 downregulated genes in EN144-EVs compared to Expi293F-EVs (|log (FC)| > 2, adj.P < 0.05; Figure 2e). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed enrichment in processes related to extracellular exosomes, protein binding, and integrin binding, suggesting minimal disruption to normal physiological functions (adj.P < 0.05; Figure 2f). Notably, 13 proteins (OPRD1, GFER, KCNC2, CYP1B1, ABTB2, PTPN13, TAS2R40, PRKN, TNF, PINK1, AHR, PPP1R9A, and JOX2) associated with cellular response to toxic substances (GO:0097237) were absent in both EV types, suggesting that EN144-EVs may have low toxicity.

The cytotoxicity of EN144-EVs was next evaluated in several cell lines (RAW 264.7, 293T, SF-MSC, and rat primary chondrocytes) using varying concentrations of EVs (ranging from 5.0 × 108 to 5.0 × 1010 particles/mL). After 24 or 48 h, LDH release, ROS generation, cell viability, apoptosis, and migration were evaluated. Several data points showed signs of cell disruption: for example, the LDH levels in 293T cells were significantly elevated at 5.0 × 108 particles/mL EN144-EVs (143% vs. 100%, P = 0.047, Supplementary Figure 8a), a modest increase in apoptosis was observed in SF-MSCs at 1.0 × 1010 particles/mL (3.88% vs. 2.90%, P = 0.004, Supplementary Figure 8b). However, these signs did not persist at higher doses (Supplementary Figure 8a, b). Furthermore, high concentrations of EN144-EVs did not significantly decrease cell viability (Supplementary Figure 8c, d) and ROS levels remained unaffected across all concentrations (Supplementary Figure 8e). Cell migration was minimally affected by EN144-EVs (Supplementary Figure 8f). Collectively, these findings suggest that intracellular uptake of EN144-EVs will not cause significant cytotoxicity to the cells we have tested.

Next, we tracked the distribution of EVs in vivo following tail vein injection. We first confirmed that EVs were fluorescently labeled following the DiR-labeling procedure, and the free DiR dye was completely removed. DiR-labeled Expi293F-EVs and EN144-EVs were injected via the tail vein into the mouse circulation system. Fluorescent imaging using the in vivo imaging system (IVIS) showed rapid accumulation of both Expi293F-EVs and EN144-EVs in the liver. Intriguingly, we observed a greater accumulation of EN144-EVs in the liver and spleen compared to Expi293F-EVs (Figure 2g, h). The reasons, however, are unknown.

An in-depth evaluation of in vivo toxicity was conducted following single intravenous administrations of varying doses of Expi293F-EVs and EN144-EVs (1.0 × 1011, 1.0 × 1010, and 1.0 × 109 particles/animal) into mice. Hematological assessments 24 hours post-treatment revealed minimal impact on blood parameters, including the levels of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), granulocytes (Gran), lymphocytes (Lymph), and monocytes (Mon). A modest elevation in RBC percentage (9.980 vs. 4.410, P = 0.046) was observed in Expi293F-EVs-treated mice compared to the PBS control group (Supplementary Figure 9a-c). Both groups’ histopathological examinations of the heart, liver, spleen, lungs, and kidneys revealed no treatment-related abnormalities (Supplementary Figure 10a). Similarly, blood biochemical markers (ALT, ALP, AST, BUN, CREA, and LDH) remained unchanged after the treatment (Supplementary Figure 9d-f). Next, we investigated the in vivo toxicity of repeated administration of EN144-EVs. Briefly, mice were dosed with the highest concentration thrice weekly (Supplementary Figure 9g). Monitoring body weight and organ coefficients revealed good overall health, with only a slight increase in the spleen coefficient in the EN144-EVs group compared to PBS (0.006 vs. 0.005, P = 0.028) (Supplementary Figure 9h, i). Subsequent hematological, biochemical, and histopathological evaluations detected no significant differences between EN144-EVs-treated and PBS-treated animals (Supplementary Figure 9j, k, Supplementary Figure 10b). Taken together, these data confirm that EN144-EVs are generally safe for systemic injections.

Surface and luminal cargo loading by EN144 engineering
Next, we installed a chondrocyte-affinity peptide (CAP, DWRVIIPPPPRPSA) on the surface of extracellular vesicles by engineering the scaffold proteins to achieve chondrocyte targeting for the treatment of osteoarthritis32. Four plasmids were constructed by fusing CAP with different EV scaffold proteins, yielding ENPP1-CAP, EN144-CAP, Lamp2b-CAP, and PTGFRN-CAP (Figure 3a). Subsequently, chondrocyte-targeting EVs, EN144-EVCAP, ENPP1-EVCAP, PTGFRN-EVCAP, and Lamp2b-EVCAP, were isolated from transfected cells. These engineered EVs showed sizes ranging from 50 to 150 nm (Supplementary Figure 11a, b). To enable direct comparison among different EVs, EV concentrations were normalized based on particle counts determined by RPS. Primary rat chondrocytes were incubated with EVs for 15 min or 2 h to evaluate cellular uptake efficiency. Quantitative analysis revealed enhanced cellular uptake of PTGFRN-EVCAP (80.38 ± 0.79), ENPP1-EVCAP (107.6 ± 19.78), and EN144-EVCAP (101.6 ± 15.32) in the 2-hour incubation group, with ENPP1-EVCAP and EN144-EVCAP exhibiting the highest fluorescence intensities (Figure 3b). Notably, after 24-hour incubation, the cellular uptake rates of ENPP1-EVCAP and EN144-EVCAP reached approximately 90%, significantly surpassing those of PTGFRN-EVCAP (87.97% ± 0.38%), Lamp2b-EVCAP (69.37% ± 0.97%), and Expi293F-EV (47.93% ± 1.27%, Figure 3c). To determine whether the observed ENPP1-EVCAP/chondrocyte interaction was mediated by CAP-specific binding, primary chondrocytes were pre-treated with CAP peptide (1, 3, 10 μg) for 2 h to block potential binding sites. Interestingly, pretreatment with 10 μg CAP peptide significantly reduced the fluorescence intensity of primary chondrocytes by 45.6% compared to untreated controls. This dose-dependent blockade indicates that CAP peptide mediates the uptake of EN144-EVCAP by primary chondrocytes (Supplementary Figure 12).

Next, we compared the loading efficiency of mRNA cargo into different EVs. We devised a two-component system leveraging the MS2 phage shell protein (MS2)-LOOP pair and the archaeal ribosomal protein (L7Ae)-C/D Box pair. MS2 is known to bind LOOP motifs, while L7Ae recognizes C/D Box sequence. By fusing EN144 with MS2 or L7Ae and inserting LOOP or C/D Box sequences into EGFP mRNA’s 3’-UTR, we achieved selective mRNA loading to EVs. A control group with mRNA expressing free, non-fused EGFP was included to provide an estimation of the random, passive mRNA encapsulation level in EVs (Figure 3d). qRT-PCR analysis revealed that compared to EVs with the MS2-LOOP pair, the EV group with the L7Ae-C/D Box pair contains the highest EGFP mRNA content (Figure 3e, Supplementary Figure 13a). Thus, we selected the L7Ae-C/D Box as a guiding cue for enriching mRNA in EVs. In addition, we compared the EGFP mRNA loading capacity of EVs with EN144, Lamp2b, and PTGFRN as the scaffold by constructing EN144-EVEGFP, Lamp2b-EVEGFP, and PTGFRN-EVEGFP. EN144-EVEGFP demonstrated the highest EGFP mRNA content compared to Lamp2b-EVEGFP and PTGFRN-EVEGFP (Figure 3f, g, Supplementary Figure 13b, c). Cellular uptake studies revealed a peak uptake at 18 h for EN144-EVEGFP, achieving a higher level of intracellular EGFP mRNA than other EV preparations (Supplementary Figure 13d, e). To determine whether the observed EGFP signal originated from active mRNA translation rather than from pre-existing protein passively carried in EVs, LC-MS/MS was used to quantify EGFP levels in three types of EVs (Expi293F-EVs, EGFP-C/D box EVs, and EN144-EVEGFP) as well as in recipient cells after co-culture (Supplementary Figure 13f, g). The results demonstrated that passive loading of EGFP protein was detected in both the EN144-EVEGFP and EGFP-C/D box EVs groups (Supplementary Figure 13f). However, EGFP expression was significantly elevated in recipient cells co-cultured with EN144-EVEGFP (Supplementary Figure 13g). These findings indicate that the EGFP signal in the recipient cells primarily resulted from active translation of EGFP mRNA delivered by EN144-EVEGFP, rather than from pre-packaged EGFP protein in the EVs. Collectively, these results demonstrate that EN144-EVEGFP, utilizing the L7Ae-C/D box system, efficiently loads EGFP mRNA in EVs and delivers the mRNA into cells.

For protein loading within the lumen of EVs, one can directly fuse the protein of interest with the scaffold through a covalent bond, or assemble the protein of interest with the scaffold protein via a specific, non-covalent interaction mediated by a pair of interacting domains. We systematically evaluated multiple protein heterodimerization strategies for EV cargo loading, including a GFP-GFP nanobody interacting pair33, a light-dependent CIBN-CRY2 interacting pair34, an NbALFAPE-ALFA interacting pair35, and a PRB-PDAR interacting pair36. We fused one binding partner, GFP, CIBN, PDAR, and NbALFAPE, with EN144, and Cas9 with the corresponding binding partner, Nanobody, CRY2, PRB, and ALFA, respectively (Figure 3h). These four plasmids were then transfected transiently into Expi293F cells. As expected, sgRNAs were detected in transfected cells and their derived EVs using qRT-PCR (Figure 3i, Supplementary Figure 14a). The qRT-PCR results clearly showed that the GFP-Nanobody system emerged as the most efficient for loading sgRNA into EVs compared to other dimerization systems (Figure 3i). Next, we evaluated the capacity of engineered EVs to deliver sgRNA into cells. The GFP-Nanobody system exhibited the greatest uptake efficiency, with intracellular levels of exogenous sgRNA exceeding those delivered by CIBN-CRY2, PRB-PDAR, and NbALFAPE-ALFA systems by approximately 4.32-, 5.57-, and 18.71-fold, respectively (Figure 3j). To further validate that Cas9 was loaded into EVs, we verified Cas9 expression in cells and secreted EVs (EN144-EVM-CRISPR/Cas9) using the GFP-Nanobody system (Supplementary Figure 14b). NTA results revealed that EN144-EVM-CRISPR/Cas9 and Expi293F-EVs had distinct diameters, which peaked at approximately 77 nm and 66 nm, respectively (Supplementary Figure 14c). To confirm the function of CRISPR/Cas9 in EVs, we first demonstrated the physical binding of sgRNA to Cas9 in EVs through RIP assays, indicating the existence of RNP complexes (Figure 3k). Subsequent co-IP analysis confirmed the loading of RNP complexes into EVs via the GFP-Nanobody system, as indicated by a Cas9 pull-down assay using an antibody against FLAG-tagged EN144 (Figure 3l).

Having established the successful loading of Cas9 into EVs via the GFP-Nanobody system, we evaluated its functional integrity. We generated a stop-dsRed reporter cell line, where a stop sequence halts dsRed gene expression. Stop-dsRed is a genetic modification that involves adding a stop sequence to the dsRed gene, effectively halting its expression. However, introducing sgRNA1 and sgRNA2 precisely cleaves this stop sequence, thereby allowing the dsRed gene expression as intended (Figure 3m). Confocal imaging confirmed the successful setup of reporter cells and plasmids, as evidenced by the expression of GFP and Red in stop-dsRed cells. Importantly, EVs containing CRISPR-Cas9 components induced detectable red fluorescent signals in stop-dsRed cells, demonstrating the function of RNP in EVs (Figure 3n). Sanger sequencing followed by TIDE (Tracking of Indels by Decomposition) analysis further revealed that the editing efficacy in the stop-dsRed gene upon EVs treatment ranged from 3.1% to 13.6% (Supplementary Table 5). These data indicate that RNP in EVs is functional in recipient cells.

Decoy EVs alleviate systemic inflammation in LPS-induced septic mouse
Cytokines IL-6 and TNF-α are key mediators of inflammatory regulation. Next, we design EVs that display cytokine receptors. Glycoprotein 130 (also known as gp130, IL6ST, IL6R-beta, or CD130) is a type I cytokine receptor that binds to IL-6. Based on previous studies37, 38, the C-terminus of EN144 was fused to mouse gp130 (mgp130), comprising the extracellular, transmembrane, and partial cytoplasmic domains, and transiently expressed in Expi293F cells. Differential ultracentrifugation yielded engineered EVs displaying EN144-mgp130 (EN144-EVmgp130, Figure 4a). RPS analysis revealed EN144-EVmgp130 diameters of 73 nm, comparable to typical EV sizes (Figure 4b). To validate the efficacy of EN144-EVmgp130, an in vitro inflammation model was established using LPS-stimulated RAW 264.7 cells. Co-culturing with 1000 ng/mL LPS for 3 h significantly upregulated TNF-α and IL-6 at both RNA (P < 0.01) and protein levels, imitating the inflammation conditions (Supplementary Figure 15a-c). Quantitative analysis revealed that EN144-EVmgp130 achieved significantly greater suppression of TNF-α and IL-6 in inflammatory RAW 264.7 cells (Figure 4c, d). Encouraged by these data, we evaluated the therapeutic potential of EN144-EVmgp130 in an LPS-induced sepsis mouse model. Septic mice exhibited heightened IL-6 and TNF-α expression in the liver, spleen, and lungs at 3 h post-LPS injection (Supplementary Figure 15d-f). EN144-EVmgp130 was found to be enriched in the liver, lungs, and spleen of septic mice 6 h after EVs were injected intravenously (Supplementary Figure 15g). Consistently, EN144-EVmgp130 treatment significantly reduced the IL-6 expression levels in the liver, lungs, and spleen (Figure 4e). In vitro and in vivo investigations demonstrated that EN144-EVmgp130 significantly attenuated IL-6 and TNF-α expression levels in a LPS-induced inflammation model.

Conservativeness analysis of gp130 across multiple species revealed high conservation levels in humans, rhesus monkeys (97.6%), rats (78.63%), and mice (77.18%), all exceeding 75%. This evolutionary conservation prompted further investigation into the therapeutic efficacy of human gp130 (hgp130). EN144-hgp130 was transfected into Expi293F cells, and hgp130 expression was detected in cells and EVs (EN144-EVhgp130, Figure 4f). Notably, despite comparable particle sizes, EN144-EVhgp130 exhibited significantly higher loading efficiency than EN144-EVmgp130 (Figure 4f, g). Similar to the therapeutic effects of EN144-EVmgp130, EN144-EVhgp130 was observed to significantly reduce both IL-6 protein and RNA levels in inflammatory cells (Figure 4h, i). Furthermore, we observed that EN144-EVhgp130 exhibit a dose-dependent inhibitory effect on IL-6. Specifically, EN144-EVhgp130 decreased IL-6 levels by 30% at a concentration of 5.0 × 108 particles/mL and by 55% at 1.0 × 1010 particles/mL (Supplementary Figure 16a). Building on these encouraging in vitro findings, the therapeutic potential of decoy EVs was subsequently evaluated in vivo using a sepsis model. Administration of varying EV doses demonstrated that intravenous injection of EN144-EVmgp130 or EN144-EVhgp130 (1.0 × 1010 particles/mouse) significantly improved survival outcomes in septic mice, achieving survival rates exceeding 80% within 120 hours post-administration (Figure 4j, k). Notably, when administered at the highest dose of 5.0 × 1010 particles/mouse, both preparations achieved complete (100% up to 120 h after injection) survival protection (Figure 4j, k). Comparative analysis revealed that EN144-EVhgp130 (80%) and EN144-EVmgp130 (80%) demonstrated an enhanced therapeutic efficacy in septic mice, with survival rates higher than those achieved by sgp130 treatment at 10 μg (40%) and 1 μg (20%) doses (Figure 4l). Moreover, mice receiving decoy EVs therapy exhibited accelerated weight recovery (72 hours) compared to the sgp130-treated group (96 hours post-treatment, Figure 4m. The decoy EVs exhibited superior protection against LPS-induced inflammation, as evidenced by reduced IL-6 levels in the liver, lung, and spleen (Supplementary Figure 16b, c). We then assessed the impact of various treatments on the histopathology of sepsis mice. Mice in the PBS group exhibited massive inflammatory cell infiltration in the liver, lungs, and spleen, and elevated histologic scores. Conversely, decoy EVs treatment mitigated inflammation in the liver, lungs and spleen, effectively alleviating LPS-induced inflammation (Supplementary Figure 16d, e). Taken together, we demonstrate the anti-inflammatory efficacy of two protein-carrying EVs using EN144 as the scaffold protein, EN144-EVmgp130 and EN144-EVhgp130, in a cell-based model and an animal model of sepsis.

To investigate the regulatory role of EN144-EVmgp130 in the IL-6 trans-signaling pathway, we first characterized its binding properties in vitro. Consistent with the canonical theory that gp130 activation requires the preformed IL-6/IL-6R complex22, EN144-EVmgp130 did not bind to IL-6 or IL-6R individually, and binding was observed exclusively with the IL-6/IL-6R complex (Supplementary Figure 17a). In control experiments, EN144-EVs showed no binding to IL-6, IL-6R, or the IL-6/IL-6R complex (Supplementary Figure 17a). These results demonstrate that EN144-EVmgp130 specifically interacts with the IL-6/IL-6R complex. Building on these findings, we evaluated the biological effects of EN144-EVmgp130 in a murine model. Treatment with EN144-EVmgp130 significantly reduced serum levels of IL-6, sIL-6R, and proinflammatory cytokines (IL-1β and TNF-α; Figure 4n-q), while decreasing the pSTAT3/STAT3 ratio in hepatic, splenic, and pulmonary tissues, collectively indicating effective suppression of IL-6 trans-signaling activation (Supplementary Figure 17b-g). Notably, expression levels of classic signaling markers—acute-phase proteins SAA and CRP—remained unaltered, excluding nonspecific interference of EN144-EVmgp130 with the canonical pathway (Figure 4r, s). These results demonstrate that EN144-EVmgp130 selectively inhibits the IL-6 trans-signaling pathway through targeted engagement of the IL-6/IL-6R complex, without appreciably affecting classical IL-6 signaling.

Chondrocyte-targeting decoy EVs to repair cartilage damage
The sepsis model evaluated the systemic administration of EN144-EVhgp130 in a non-targeted way. Next, we seek to assess the anti-inflammatory efficacy of EN144-EVhgp130 for targeted delivery of protein to chondrocytes in the cartilage as a potential treatment for OA. Cartilage is a dense tissue that is difficult to penetrate. A CAP sequence was attached to the C-terminus of EN144-hgp130, resulting in EN144-hgp130-CAP. Subsequently, the plasmid was transiently transfected into Expi293F cells, and EN144-EVhgp130-CAP were isolated by ultracentrifugation. Based on the FLAG tag fused to the C terminus of the EN144-hgp130 sequence, we detected the fusion protein in all the transfected cells and EVs, and no notable change in EV sizes was observed (Figure 5a, Supplementary Figure 18a, b). The chondrocyte-targeting capacity of engineered EVs was evaluated using DiI-labeled EVs co-cultured with primary chondrocytes. Quantitative analysis by confocal microscopy demonstrated significantly enhanced binding of EN144-EVhgp130-CAP to chondrocytes (115.4 ± 10.77) compared to EN144-EVhgp130 (86.79 ± 0.12) at 2-hour post-incubation (Figure 5b). Prolonged 24-hour co-culture revealed superior internalization efficiency of EN144-EVhgp130-CAP, with 95.33% ± 0.31% DiI-positive cells versus 62.90% ± 0.66% in the EN144-EVhgp130 control group (Figure 5c). Moreover, intra-articular injections of EN144-EVhgp130-CAP demonstrated higher fluorescence intensity in the joints at 24 and 168 hours than the group treated with EN144-EVhgp130, and minimal systemic distribution to other organs (Supplementary Figure 18c). These data show that chondrocyte-targeting EN144-EVhgp130-CAP had significantly higher cellular uptake in vitro and cartilage-specific retention in vivo.

After validating the chondrocyte-targeting capability, we next investigated the anti-inflammatory efficacy of EN144-EVhgp130-CAP in chondrocytes. EN144-EVhgp130 or EN144-EVhgp130-CAP significantly promoted the proliferation and migration of inflammatory chondrocytes and reduced apoptosis compared with other treatment groups (Figure 5d-h, Supplementary Figure 19a-c). Notably, the CAP peptide on the surface of EN144-EVhgp130-CAP delivered hgp130 more efficiently into inflammatory chondrocytes, promoting chondrocyte repair more effectively than EN144-EVhgp130 (Figure 5d-h, Supplementary Figure 19a-c), highlighting the potential advantage of the targeting effect. The above results indicated that EN144-EVhgp130-CAP exerts enhanced chondroprotective effects compared to other treatments on inflammatory chondrocytes, thereby confirming the high anti-inflammatory efficacy of EN144-EVhgp130-CAP in vitro.

The efficacy of EN144-EVhgp130-CAP in vivo for cartilage repair and potential OA treatment was evaluated. OA conditions were induced in rats using the ACLT surgical technique. The rats were divided into groups receiving intra-articular injections of various substances, including PBS, hyaluronic acid (HA), EN144-EVs, EN144-EVhgp130, or EN144-EVhgp130-CAP. A control group undergoing a sham surgery was also included in the study. The injections were administered once a week for four consecutive weeks, and rats were euthanized in the fifth week for further analysis (Figure 5i). Based on the OA Research Society International (OARSI) score, the EN144-EVhgp130-CAP treatment group exhibited the best recovery, as indicated by the lowest score. Comparing the EN144-EVhgp130-CAP treatment group and the EN144-EVhgp130 group, the targeting property of EVs provided a more pronounced chondroprotective effect (Figure 5j).

Next, we conducted immunohistological staining to verify the therapeutic efficacy. HE, SO-FG, and TB staining were performed to assess cartilage degeneration in each group. The ACLT group exhibited cartilage thinning and decrease proteoglycan levels, whereas HA and EN144-EVhgp130 treatment partially ameliorated these features (Figure 5k). The EN144-EVhgp130-CAP treatment group showed the most pronounced alleviation of cartilage degeneration and abrasion in OA rats (Figure 5k). IHC analysis revealed elevated COL2A1 and reduced IL-6 and MMP13 expression in the treatment groups compared to the ACLT group. Notably, the EN144-EVhgp130-CAP group showed the highest COL2A1 level and the lowest levels of IL-6 and MMP13, which were superior to the levels in the EN144-EVhgp130 and HA groups (Figure 5l-o). These results indicate that EN144-EVhgp130 and EN144-EVhgp130-CAP groups promoted cartilage repair, with the EN144-EVhgp130-CAP treatment being the most effective.

OA is characterized as a chronic inflammatory condition driven primarily by the actions of innate immune cells, particularly macrophages located in the synovial tissue. Immunofluorescence (IF) imaging of the synovium revealed an elevated presence of M1-like macrophages (iNOS+) and a reduced proportion of M2-like macrophages (CD206+) in OA rats. Quantitative analysis demonstrated a higher percentage of M2-like macrophages in all treatment groups than in the PBS group (Supplementary Figure 20a, c, d). Notably, treatment with EN144-EVhgp130-CAP resulted in decreased IL-6 expression in synovial tissue, comparable to the Sham group (Supplementary Figure 20b, e). To evaluate the safety of intra-articular injection of EN144-EVhgp130-CAP in vivo, rat organs (heart, liver, spleen, lungs, kidneys, and blood) were collected post-treatment. No significant pathological changes were observed in the EN144-EVhgp130-CAP treatment group compared to the Sham group (Supplementary Figure 21a-g). These results suggest that the EN144-EVhgp130-CAP treatment effectively alleviates the OA inflammation by promoting the polarization of M2-like macrophages. Therefore, EN144-EVhgp130-CAP improves the microenvironment of synovial tissue, reduces cartilage tissue inflammation, and thereby promotes cartilage tissue regeneration.

Methods
Materials and reagents
Expi293F cells (#A14528), Penicillin-Streptomycin (#15070063), Lipofectamine 2000 (#1668019), and Protein A/G Magnetic Beads (#88802) were acquired from Thermo Fisher Scientific, MA, USA. RAW 264.7 cells (#KGG2201-1), 293T cells (#KGG3101-1), and dihydroethidium (DHE) probe (#KGA7502-5) were provided by keyGEN Biotech Inc., China. Fetal Bovine Serum (FBS, #SA112.02) was sourced from CellMax, China. Additionally, Dulbecco’s Modified Eagle’s Medium (DMEM, #BL304A) and Phosphate Buffered Saline (PBS, #BL302A) were acquired from Biosharp, China. The Rat IL-1β protein (#80023-RNAE), and Mouse gp130/IL6ST protein (50135-M02H) were purchased from SinoBiological, China. The Mouse IL-6 protein (#CG39), and Mouse IL-6RA protein (#C06P) were acquired from Nonoprotein, China. The ELISA kit used to detect CRP (#YM-2810A1), sIL-6R (#YM-6368A1), SAA (#YM-3116A1), IL-6 (#YM-2899A1), IL-1β (#YM-2776A1), and TNF-α (#YM-2868A1) were provided by YOUMENG BIOLOGY, China. Albumin Bovine (BSA, #9048-46-8) and collagenase II (#9001-12-1) were acquired from BioFroxx, Germany. The PVDF membranes (#ISEQ00010), 0.22 μm filter membranes (#SLGPR33RB), and Lipopolysaccharide (LPS, #L2630) were purchased from Sigma-Aldrich, MO, USA. Chemiluminescence kit (#E422-02-AB), FreeZol Reagent Kit (#R711-02), Prestained Protein Marker (#MP102-02), and SYBR Green PCR Mix (#Q712–02) were obtained from Vazyme Biotech Co., Ltd., China. The DiR (#C019) DiI dyes (#C017), and Hochest dyes (#FP027) were purchased from ABP Biosciences, MD, USA. Matrigel™ Basement Membrane Matrix (#356234) was provided by BD Biosciences, CA, USA. Hyaluronic acid (HA, #9067- 32-7, Molecular Weight ≥ 1.8 MDa, intrinsic viscosity ≥27.2 DL/G) were obtained from Aladdin, China. The DAB chromogen Kit (#GS1212-200T) was obtained from Servicebio, China. PEI MW40000 (#40816ES), 2 × Hieff® PCR Master Mix (#10102ES03), Annexin V-YSFluorTM (#40304ES60) were purchased from Yeasen, China. LDH Cytotoxicity Assay Kit (#C0017), Antifade Mounting Medium with DAPI (#P0131), PMSF (#ST2573), Protease inhibitor cocktail for general use (#P1005), RNase inhibitor (#R0102), Proteinase K (#ST535) were provided by Beyotime, China. Evtrap (EV02-01-01) was obtained from EVLiXiR, China. A comprehensive list of antibodies used in this study is detailed in Supplementary Table 1.

Cell culture
The cell lines RAW 264.7, 293T, HcerEpic, HeLa, Rat Primary Chondrocytes (Chondrocytes) and synovial fluid-derived mesenchymal stem cells (SF-MSC) were maintained in DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin. SF-MSCs were a gift from Dr. Yujie Liang67. Chondrocytes were extracted according to a previous protocol68. Expi293F cells were cultured in HEK293 Media under continuous shaking at 120 rpm. All cell types were housed in humidified incubators at 37°C and 5% CO2.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
LC-MS/MS processing and analysis were performed as previously described32. The peptide samples were subjected to analysis using the QE HF-X instrument (Thermo Fisher Science, USA). For the proteomic analysis, EVs lysates (3.0 × 109) underwent chromatographic separation on the nLC 1200 system (Thermo Scientific, USA), equipped with a C18 chromatographic column (2.2 µm, 100 Å; Michrom Bioresources). The chromatographic separation was performed at a flow rate of 300 nL/min, utilizing an aqueous solution of 0.1% formic acid (solvent A) and an 80% acetonitrile solution containing 0.1% formic acid (solvent B) as the mobile phases.

The chromatographic column was initially equilibrated with 100% of liquid A, followed by a gradient elution: 2-8% B for 0-3 min, 8-40% B for 3-81 min, 40-95% B for 81-83 min, and maintained at 95% B for 83-90 min. Data acquisition was performed in data dependent acquisition (DDA) mode, with MS1 settings of 60,000 resolution, AGC target of 3e6, maximum inject time of 30 ms, and a scan range of 400-1200 m/z. For MS2, the settings included 30,000 resolution, AGC target of 1e5, maximum inject time of 50 ms, loop count of 20, collision energy of 28, and dynamic exclusion of 40 s.

The raw data were searched using Proteome Discoverer 2.3 software, configured with trypsin/P as the enzyme, allowing up to 3 missed cleavages. Fixed modifications included carbamoyl methylation (+57.02 Da), while variable modifications encompassed methionine oxidation (+15.99 Da) and acetylation (+42.01 Da). Both protein and peptide false discovery rates (FDRs) were set to 1% to ensure high data quality.

To detect the expression of EGFP in engineered EVs, EVs lysates derived from a standardized input of 3.0 × 109 particles (as determined by Resistive Pulse Sensing) underwent proteomic analysis. Label-free quantification (LFQ) of EGFP was performed using PEAKS Online with the following parameters: database, UniProt human proteome + EGFP sequence; enzymatic specificity, set to trypsin/P, and allowed up to 2 missed cleavages; fixed modification, carbamidomethylation (+57.02 Da); variable modification, methionine oxidation (+15.99 Da) and acetylation (+42.01 Da); FDR, protein and peptide false discovery rates set to 1%. EGFP abundance was normalized to total protein intensity per sample and reported as LFQ intensity. The raw LFQ intensity for EGFP was used for cross-sample comparison. To account for potential minor technical variations during mass spectrometry runs, the data was also processed with a total protein intensity normalization in the PEAKS software; however, all biological interpretations are made based on the comparison of signals originating from an equal number of EV particles.

Transfection and stable cell line selection
To generate stable cell lines, the target plasmids were transfected into Expi293F cells using electroporation (Celetrix, USA) or PEI MW40000 transfection reagents. Specifically, Expi293F cells (1.0 × 107) were mixed with the plasmid (20 µg) and transferred to a 200 µL electro-transfer tube. Cell Line mode was then selected, and a single electro transformation at 1125 V was performed. Unlike electroporation, PEI transfection was performed by mixing the plasmid (20 µg) and PEI MW40000 transfection reagent (1 mg/mL, 60 µL) for 15 min and then adding it uniformly dropwise to the medium containing Expi293F cells (1.0 × 107). Stable cell lines were screened by G418 and regular passaging until the viability of the cell lines was restored to more than 90%, and then detected the expression of target genes6.

293T, HcerEpic, HeLa, and SF-MSC cells were seeded in 100 mm culture dishes (1-2 × 106 cells/dish) 24 hours prior to transfection. Cells at 70-80% confluency were transfected with 10 μg of EN144 plasmid using 20 μL PEI MW40000 via incubation of DNA-PEI complexes (formed in serum-free DMEM, 15 min RT) followed by dropwise addition; complete medium replacement was performed at 6 h post-transfection. Conditioned media were collected at 48 h and 72 h for EVs isolation.

EVs isolation and characterization
According to the previously established protocols, EVs were extracted and characterized, and the viability of cells before EVs isolation was maintained above 95%28. Morphological analysis was conducted using TEM (HITACHI, Japan), where EVs were deposited onto Formvar-coated nickel grids, negatively stained with 2% uranyl acetate, air-dried, and visualized. Resistive Pulse Sensing (RPS) was employed for the determination of particle size and concentration. Using a NanoCoulter counter (Resun Technology, China), a chip with a measurement range of 60 nm to 200 nm was installed, and 200 µL of PBS buffer was rapidly dispensed into each sample well on both sides of the detection card, ensuring the system was free of air bubbles or leaks. Before sample analysis, a background verification was performed, confirming that the particle count in PBS was fewer than 5 over 60 seconds. In each sample, 100 EV particles were selected for size analysis. To detect marker proteins in EVs, Western blot (WB) analysis was performed to detect the expression levels of TSG101, Hsp70, CD9, and Calnexin in cells or EVs. First, the lysed cells (10 µg) or EVs (3.0 × 109) proteins were quantified by NanoDrop (Thermo Scientific, USA), and then the proteins were separated by 8%-15% SDS-PAGE. Subsequently, the proteins were transferred to 0.22 μm or 0.45 μm PVDF membranes and closed with 1% BSA. Next, incubation of primary and secondary antibodies was performed, and finally, an enhanced chemiluminescence solution was used to show protein bands.

Plasmid design and construction
All protein sequences were obtained from UniProt to generate chimeric proteins (protein sequences available in Supplementary Table 2). During the plasmid construction process, the following elements were utilized: the FLAG tag (DYKDDDDK), a general signal peptide (MGWSCIILFLVATATGVHS), a protective peptide (GNSTM), a translation-promoting Kozak sequence (GCCGCCACC), and a flexible linker peptide (GSGS). The sorting protein sequence with N-terminal located in the EVs lumen was EGFP-flexible peptide-FLAG tag protein-sorting protein-FLAG tag protein, and this sequence was constructed in the pEGFP-C1 vector. The sorting proteins with C-terminal located in the EVs lumen had the following sequence: promoting translation peptide-universal signal peptide-protective peptide-FLAG tag protein-Sorting protein-FLAG tag protein-flexible peptide-EGFP, and this sequence was constructed in pCDNA3.1 vector. The PDGFR sequence was incorporated into pDisplay plasmid, enabling EGFP fusion to PDGFR within plasmid. Expression was initiated by the CMV promoter on the carrier, with EGFP labels on the carrier protein and G418 resistance genes used for detection and screening of positive cells.

Cellular uptake of EVs
To evaluate the targeting efficiency of EVs, glass coverslips were placed at the bottom of 12-well plates, and each well was seeded with 8.0 × 104 cells and cultured overnight. 3.0 × 109 counts of DiI-labeled EVs were added to each well and co-cultured with cells for 15 min or 2 hours. Subsequently, the medium was aspirated, and cells were washed twice with PBS. Cells were then fixed using 200 µL of 4% paraformaldehyde (PFA) for 20 min at room temperature. After washing, cells were further incubated with 500 µL serum-free DMEM medium containing Hoechst 33342 at 37°C for 30 min to label nuclei. The slide was then sealed with an anti-quenching and visualized under a confocal laser microscope (Olympus, FV1000) with the following excitation/emission settings: DiI (549 nm/565 nm), Hoechst (350 nm/461 nm), and EGFP (488 nm/507 nm).

LDH cytotoxicity assay, cell proliferation, apoptosis, migration assay, and reactive oxygen species (ROS)
To assess the cellular impacts of varying EN144-EVs concentrations, a series of cytotoxicity and phenotyping experiments was employed. Prior studies have established the non-cytotoxic nature of Expi293F-EVs across a concentration (range 5.0 × 108 particles/mL to 5.0 × 109 particles/mL) in HepG2 cells, prompting our investigation into the cytotoxicity of EN144-EVs at concentrations (including 5.0 × 108, 1.0 × 109, 5.0 × 109, 1.0 × 1010, and 5.0 × 1010 particles/mL) on RAW 264.7, 293T, Chondrocytes, and SF-MSCs.

For the LDH assay, cells were seeded at 5,000 cells/well in 96-well plate and incubated overnight. Following 24 h exposure to various EN144-EVs concentrations, cells were centrifuged at 400 g for 5 min, and the supernatant, post-LDH release reagent treatment, was analyzed after further centrifugation. The resulting supernatant (120 µL) was transferred to a new plate and mixed with LDH assay solution (60 µL), incubated at room temperature for 30 min, and absorbance measured at 450 nm to quantify LDH release.

For the level of ROS on cells by EN144-EVs, DHE probe was employed. DHE is a marker of intracellular peroxides, and once oxidized, it enters the DNA of the cell nucleus, allowing the nucleus to emit a bright red fluorescence. After the cells were exposed to different concentrations of EN144-EVs for 24 h, the medium was replaced with fresh medium (1 mL/well) and DHE probe (1 µL/well) was added. After incubation for 30 min in the dark, the proportion of fluorescent cells was quantified by flow cytometry.

The effects of EN144-EVs on cell proliferation, apoptosis, and migration were evaluated using standardized protocols for CCK-8, flow cytometry, scratch, and transwell assays, respectively, consistent with previous publications28, 69.

Quantitative real-time PCR (qRT-PCR)
To elucidate the impact of various treatments on gene expression profiles, qRT-PCR was conducted. Total RNA was efficiently extracted from EVs, cells, and tissues using the FreeZol Reagent, adhering strictly to the manufacturer's procedure. This RNA was then converted to cDNA through reverse transcription utilizing the RT MasterMix Kit. Subsequently, qRT-PCR was performed with SYBR Green PCR Mix for relative quantification. The 2-△△CT method facilitated the analysis of results. The primers relevant to the study were listed in Supplementary Table 3.

RNA immunoprecipitation (RIP) assay
Cells and EVs were lysed in a buffer containing 10 mM Tris, 200 mM NaCl, 30 mM EDTA, 0.5% Triton-X 100, 0.5% NP40, 1 mM PMSF, 1% protease inhibitor cocktail, and 10 U/mL RNase inhibitor for 30 min on ice. Following centrifugation at 16,000g for 10 min at 4°C, anti-Cas9 antibody or IgG was incubated with 50 μL Protein A/G Magnetic Beads for 30 min at 4°C. Overnight incubation at 4°C ensued with the supernatants and beads. Subsequently, immunoprecipitated complexes were eluted from the beads using a lysis buffer. These complexes were then processed for either western blot analysis using RIPA buffer or RNA analysis using FreeZol Reagent.

Co-Immunoprecipitation (co-IP)
Lysed cells and EVs were supplemented with either 3 μg of anti-FLAG antibody or anti-IgG antibody and incubated overnight at 4℃. Subsequently, 50 μL of Protein A/G Magnetic Beads were added to the mixture and incubated at room temperature for 2 h. Unbound complexes were thoroughly removed through two wash steps using a total of 500 μL of wash buffer. The antigen-antibody complexes were then eluted with lysis buffer and further processed with RIPA buffer for WB analysis.

Sanger sequencing
Sanger sequencing was employed to assess the editing efficiency at the target genomic loci. PCR amplification was performed in a 20 µL reaction mixture containing 3 µL of template DNA, 1 µL each of forward and reverse primers (10 μM), 10 µL of 2 × Hieff® PCR Master Mix, and 5 µL of ddH₂O. The amplification protocol consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 25 s, annealing at 62°C for 30 s, and extension at 72°C for 20 s, with a final hold at 4°C. The resulting DNA fragments were separated by electrophoresis on a 2% agarose gel. Subsequently, the purified PCR products were subjected to Sanger sequencing, and the editing efficacy was analyzed using the TIDE algorithm70. Editing efficiency was determined by sequencing analysis of the target locus in genomic DNA extracted from the FACS-sorted, red fluorescent-positive cell population. It represents the frequency of indel mutations among all stop-dsRed copies in the fluorescent 293T cells.

Enzyme-linked immunosorbent assay
Serum levels of IL-6, soluble IL-6 receptor (sIL-6R), C-reactive protein (CRP), serum amyloid A (SAA), TNF-α, and IL-1β were quantified using a two-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well microplates were pre-coated with capture antibodies specific to each target protein. Standards (recombinant proteins) and serum samples were loaded into designated wells, followed by incubation with horseradish peroxidase (HRP)-conjugated detection antibodies. After sequential incubation and thorough washing, tetramethylbenzidine (TMB) substrate was added for chromogenic development. The enzymatic reaction was terminated by adding a stop solution, converting the blue chromogen into a stable yellow product. Optical density (OD) was measured at 450 nm using a microplate reader, and analyte concentrations in mouse serum were calculated based on standard curves.

In Vivo Distribution of EVs
EVs were labeled with the near-infrared fluorescent dye DiR (10 mM) by incubating 1.0 × 1011 particles/mL EVs with 5 µL of DiR dye at 37°C for 30 min. Unbound dye was removed using a 0.22 µm filter, and the DiR-labeled EVs were resuspended in sterile PBS. Mice or rats received either tail vein or intra-articular injections of DiR-EVs. At designated time points post-injection, animals were anesthetized and transferred to an IVIS Spectrum imaging system (PerkinElmer). Fluorescent signals were acquired at excitation/emission wavelengths of 754/778 nm. For ex vivo tissue analysis, euthanized animals were dissected, and major organs (e.g., heart, liver, spleen, lung, kidneys, legs) were placed on black cardboard for fluorescence imaging under identical optical settings.

Histopathological examination and Immunofluorescence staining (IF)
Histopathological examination and IF were performed to assess the therapeutic effect of EVs, as described in previously28. Briefly, after the sample is embedded and trimmed, it is sliced. The sections are then sequentially immersed in xylene for 15 min, followed by another 15 min in xylene, and then in absolute ethanol I and II for 2 min each. Finally, they are immersed in 95% ethanol, 70% ethanol, and double-distilled water for 2 min each to complete the dewaxing process. Sections were stained with hematoxylin and eosin (HE), toluid and eosin (HE), toluidine blue (TB), and Safranin O fast green (SO-FG). For immunohistochemistry (IHC), paraffin-embedded sections were stained with anti-MMP13, anti-IL-6 or anti-COL2A1, followed by staining using the DAB chromogen kit. In the immunofluorescence experiment, tissue sections were first subjected to antigen retrieval and blocking procedures. Subsequently, primary antibodies (iNOS and CD206) were added and incubated at 4°C for 24 h. Following this, the sections were washed in PBS solution, and fluorescent secondary antibodies were applied and incubated in the dark at room temperature for 1 h. Finally, DAPI staining was performed, and the sections were blocked.

In vivo toxicity assay
To detect EN144-EVs toxicity in vivo, male C57BL/6 mice (18-24 g) were randomly administrated intravenous PBS, Expi293F-EVs, and EN144-EVs. Based on treatment protocols from clinical studies involving COVID-19 patients, each patient received an intravenous injection of 3.26 × 1011 amniotic fluid-derived EVs71. Considering a human body weight of 70 kg and a mouse body weight ranging from 18-24 g, along with an extrapolation factor of 9.1 between mice and humans, the calculated dosage for each mouse is approximately 0.763-1.02 × 109 particles EVs. To assess the toxicity of EN144-EVs administered intravenously within a 100-fold dosage range, three groups were established: a high-dose group receiving 1.0 × 1011 particles/mouse, a mid-dose group receiving 1.0 × 1010 particles/mouse, and a low-dose group receiving 1.0 × 109 particles/mouse. Each group consisted of 6 mice, and 24 h post-injection, we collected tissues and blood samples from the mice for toxicity evaluation.

The high-dose EN144-EVs were injected three times weekly for one week to evaluate the toxicity of repeated administration of EN144-EVs. The body weight of mice was measured from day 1 to day 6 following the initial EN144-EVs injection. Whole blood samples were collected for routine and biochemical analyses. Subsequently, samples of the heart, liver, spleen, lungs, and kidneys were collected, fixed in 4% paraformaldehyde, and embedded in paraffin for subsequent histological experiments.

Animal housing conditions
C57BL/6 mice and SD rats were housed in the Specific Pathogen-Free (SPF) barrier facility at the Animal Center of Southeast University. Under the supervision of the Animal Ethics Committee, all animals were co-housed under strictly controlled environmental conditions, including a 12-hour light/dark cycle, a temperature of 22 ± 2°C, and a humidity of 50 ± 10%, to minimize variation. The facility maintained sterility through the use of disinfected equipment, autoclaved feed and water, and strict air-shower entry protocols.

Animal models
The LPS was used to induce a sepsis model, and male C57BL/6 mice (18-22 g) were selected for the experiment. Each mouse was given 10 mg/kg of LPS via intraperitoneal injection. After LPS induction, different drugs were administered through tail vein injection, and the time of death and changes in body weight of the mice were recorded.

To establish an animal model of OA, male SD rats weighing 190-210 g were subjected to anterior cruciate ligament transection (ACLT) as previously described. Briefly, the rats were anesthetized with 3% isoflurane. For the sham surgery (control group in this study), both knees were exposed after a medial capsule incision of the patellar tendon, and the skin incision was closed with sutures. For ACLT, the ACL of both knees was cut after opening the joint capsule. The skin incision was closed after tissue debris was removed by rinsing with saline. Four weeks after ACLT, the rats were randomly divided into five groups (n = 5 rats/group) receiving different treatments: PBS, HA (the widely clinically used injectable conservative drug for OA), EN144-EVs, EN144-EVhgp130, and EN144-EVhgp130-CAP. The rats received intra-articular injections through the joint cavity once a week for 4 weeks and were sacrificed in the fifth week to collect cartilage and internal organs for histological analysis.

Study approval
All animal experiments were authorized by the Animal Ethics Committee of Southeast University under reference number 20220301031.

Statistics and reproducibility
Unless otherwise specified, all experiments were performed in three independent replicates, and the displayed Western blots, gels, and fluorescence images were selected as representative images from three independent replicate experiments. Statistical evaluations were conducted utilizing GraphPad Prism version 8.02 (GraphPad Software, USA). Two-tailed t-tests assessed statistical significance between two groups with parametric data. For comparisons among multiple groups, either one-way ANOVA or Brown-Forsythe ANOVA test was used, followed by Dunnett’s or Dunnett’s T3 multiple comparisons test, respectively. P < 0.05 was deemed statistically significant. Data are presented as mean ± standard deviation (SD).

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD057965 (https://www.ebi.ac.uk/pride/archive/projects/PXD057965). Source data is available for Figures 1b-1d, 1g, 2a, 2b, 2e-2h, 3b, 3c, 3e-3g, 3i-3l, 4a, 4c-4f, 4h-4s, 5a-5o, and Supplementary Figures S1c, S3, S4, S5, S6, S7, S8, S9, S11, S12, S13, S14, S15, S16, S17, S18, S20, and S21 in the associated source data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes a discovery-based project and genetic engineering of cells to endogenously express an EV sorting protein/ scaffold. The authors claim that when EVs possess this novel sorting protein, the EVs have enhanced ability to target a cell (when co-expressed with a specific cell targeting protein) and load therapeutic cargo. Based on proteomic studies, the authors purport that a sorting protein, ENPP1, is superior to other sorting proteins previously identified, and that a truncated 144 aa ENPP1mversion (EN-144) is even more optimal. The authors then tested the EN-144 expressing EV in several paradigms. The author tests the EN-144 in collaboration with gp130, acts as a decoy in IL-6/IL-6 receptor system in models of sepsis and osteoarthritis, a degenerative disorder that is highly inflammatory. Cell targeting proteins (CAP) are employed in EVs that target chondrocytes in the osteoarthritis model. A weakness is that the studies to prove targeting are not rigorous. The authors conclude that then EN-144 engineered EVs can target chondrocytes and ultimately antagonize inflammation. While the concept of the project is exciting and thoughtful in regard to developing a new therapeutic, the manuscript is very dense and lacks sufficient details and controls in several experiments that dampens this reviewer’s enthusiasm for the work.\nOverall Strengths\n• The authors used 3 distinct methods to isolate EVs which adds to rigor of this EV study and strengthens their mass spec results.\n• Discovery approach - performed comprehensive proteomic analyses for sorting proteins that might improve the current standard.\n• Identifying mechanisms that can assist with targeted drug delivery by EV have great therapeutic potential is many diseases, not just inflammation\n• In Vivo studies designed to test the safety of the EN-144 EVs\n• Used two distinct models for biological efficacy – sepsis and osteoarthritis\nFocus on GP130 that has a broader range than TNFR1\nWeaknesses\n• It is odd that whole cell lysates of EXpi293F do not express CD9 (Fig. 1S) and that CD9 is only observed in the EVs.\n• In Figure 2, the GAPDH immunoblotting is not consistent and difficult to interpret.\n• In Figure 4, the timing of the EV uptake is not clear; was live cell imaging employed? There are no clear methods included for the EV uptake studies.\n• The Lamp2bEV high powered image lacks focus compared to the other images. Thus, it is very difficult to make quantitative comparisons.\n• Since EVs are heterogenous, the authors results are likely not consistent for all EVs derived from various sources.\n• The expression levels of TNFa and IL-6 are not reported in pg/mL or in quantitative units. It’s not clear from the graph if the data are fold increase or decrease. The concentration of the cytokines should be revealed\n• Binding of IL-6 or TNFa to the EN144-EV is not shown. The evidence for being a “decoy” relies on reduced inflammatory mediators in the liver and spleen. Binding kinetics of the EVs to specific inflammatory cytokines, like IL-6, should be shown.\n• The uptake studies of EV-144-cap are not convincing. Cellular uptake of EVs in 24 hours is a very late timepoint for measuring EV. The methods for targeting and uptake are not rigorous.\n• Other cell markers, not just calnexin should be used to confirm the lac of cellular debris. What was the viability of the cultured cells prior to exosomes isolation?\n• Figure 6C shows low magnification images only. Higher magnification should be included.", "reviewer_2": "The manuscript submitted by Yan et al. describes a 144-amino-acid scaffold protein for extracellular vesicle (EV) surface engineering. While the topic is highly relevant, the study suffers from poor experimental methodology, overstatement of results, and a lack of proper controls, leading to a misleading presentation of the platform’s potential. In its current format, this paper cannot be published. I would consider the manuscript for publication only if all points below are adequately addressed.\n- Figure 1D lacks clarity regarding mass spectrometry-based quantification. The method used is not specified, nor is it explained how data were normalized or how proteins per single EV were estimated. Without these details, it is difficult to assess the validity of these findings.\n- Stable cell line characterization is inadequate. There is no explanation of how the authors ensured that gene insertion copy numbers were consistent across cells, which could introduce a major bias in assessing EV cargo loading.\n- Results presented in Figure 1H and 1I are contradictory. The Western blot shows negligible expression of EN52 and EN596 in cells, yet the authors claim high EV loading efficiency. This suggests that variability in expression levels, rather than scaffold efficiency, may be the underlying cause.\n-The truncation study lacks a clear rationale. ENPP1, ENP144, and ENP596 contain the same functional domains, yet the authors claim they behave differently without providing an adequate structural or mechanistic explanation.\n- Rab7 is incorrectly used as a membrane display construct. It is a luminal protein, making its selection as a membrane-bound protein highly questionable.\n- The study does not assess whether ENP144 works across different cell types or is limited to Expi293T cells. A broader range of cell lines should be tested to evaluate its generalizability.\n- The toxicity study is poorly controlled. There is no benchmarking against unengineered Expi293 EVs, making it impossible to determine whether the observed bell-shaped viability effect is specific to ENP144-engineered EVs.\n- The biodistribution study is severely limited by small sample size. Signal quantification should also be presented graphically.\n- The increase in RBC count following high-dose EV injection is unexplored. The authors should discuss whether this is an artifact or a physiologically relevant observation.\n- Figure 4B-C does not support the claim that EN144 and Emp11 EVs have significantly higher binding affinity than PTGFRN and Lamp2b. The text should be revised to accurately reflect the results.\n- Figures 4J and 4N contain overstated claims about delivery efficiency. Editing efficiency must be quantified by sequencing to establish actual percentage editing rates. The authors should also clarify whether they used VSVG to enhance endosomal escape, as EVs inherently are poor at performing endosomal escape.\n- The orientation of displayed receptors is not addressed. ENPP1 is a type II transmembrane protein, whereas TNFR and IL6ST are type I transmembrane proteins. The effect of this difference on receptor functionality is completely ignored.\n- Figure 5 does not convincingly demonstrate a decrease in IL-6 and TNF-α levels. TNF-α levels remain unchanged or even increase following treatment with TNF decoy EVs, contradicting the authors' claims.\n- No dose-response study has been conducted for the experiments in Figure 5, this is important to perform in order to determine the true efficacy of the treatment.\n- The biodistribution study in Supplementary Figure 11H is based on a single animal and cannot be used to support conclusions.\n- Figure 5M does not clarify the differences in effects between mouse GP130 EVs and human GP130 EVs, despite both performing similarly in suppressing IL-6 trans-signaling.\n- Supplementary Figure 9 does not explain how translation activity was measured. The method must be described in detail to prevent misinterpretation of the results.", "reviewer_3": "The manuscript „Identification of a 144-amino-acid Scaffold Protein for Extracellular Vesicle Engineering” by Wenjing Yan and colleagues presents a novel strategy to generate engineered extracellular vesicles (EVs). Using an unbiased mass-spectrometry-based proteomic approach, they identify fifteen candidate proteins that could potentially be used to engineer EVs. They then focus on ENPP1 and show that a truncated version of the protein consisting only of 114 amino-acid residues is sufficient to load EVs with peptides, proteins and even mRNAs. Afterwards, they use this novel technique to engineer decoy EVs that present the extracellular portion of the signal-transducing receptor gp130 on their surface in order to inhibit the IL-6 trans-signaling pathway. They evaluate their novel EVs in two animal models (LPS-induced sepsis in mice and a rat model of osteoarthritis) and show that their EVs are able to reduce inflammation in these models, suggesting that gp130-decorated EVs can potentially be used as novel anti-inflammatory therapeutics.\nThe manuscript contains a high amount of data and consists basically of two different parts: First, the development of a novel method to generate EVs based on the truncated ENPP1 and second, the use of this novel method to generate an inhibitor that blocks IL-6 trans-signaling. While the first part is well done in my opinion, the second part is not and lacks important experiments and appropriate controls. Furthermore, the authors use tocilizumab in their rodent models, an antibody that blocks the human IL-6 receptor, but is known to have no activity in mice and rats.\nMajor points:\n1. The authors claim that decoration of their EVs with gp130 makes this an inhibitor of IL-6 trans-signaling. This would mean that IL-6 in complex with the soluble IL-6R would bind to the gp130 on the EVs, resulting in inactivation. IL-6 alone would not bind to the EVs. However, none of these things is shown in the manuscript; only indirect evidence (reduction of IL-6 in organs and tissues) and reduced inflammation based on staining of organ sections is shown. Please provide evidence that i) EVs with gp130 bind IL-6/sIL-6R complexes, but not IL-6 or sIL-6R alone, ii) measure serum levels of IL-6 and sIL-6R in vivo and demonstrate that sufficient EVs are present to neutralize IL-6 trans-signaling in vivo, iii) provide evidence (by measuring acute phase proteins) that classic signaling is still intact in vivo and only IL-6 trans-signaling is blocked.\n2. It is known from the literature that monomeric gp130 is a rather weak inhibitor of IL-6 trans-signaling. Dimerization of gp130 increases the affinity towards IL-6/sIL-6R significantly. Do the EVs express monomeric or dimeric gp130 on their surface?\n3. The authors use tocilizumab in their rodent models. Tocilizumab does not block the IL-6R in the mouse and does not block the IL-6R in the rat, it only works in humans. I provide several papers in the specific comments that prove this. For rodent studies, other anti-IL-6R antibodies are available that work in these species. Thus, the in vivo experiments as done by the authors are worthless, unfortunately.\nGeneral comments:\n• Quality of the western blots is insufficient and has to be improved.\n• Quality of the figures has to be improved.\nFurther specific comments:\n• Line 104: Etanercept is not a monocloncal antibody, but rather a designer protein consisting of the extracellular part of TNFR2 and the constant part of a IgG antibody\n• Line 109: The canonical term is “classic signaling”, not “cis-signaling”\n• Lines 119/120: #22 is not the correct citation for this statement. The correct citation is Jostock et al. Eur J Biochem. 2001 Jan;268(1):160-7\n• Figure 1G: “domin” should probably be “domain” and “cataytic” should be “catalytic”\n• For some Western blots molecular weight markers are indicated, while this is missing for others. Please add them to all western blots\n• Original, uncropped blots are currently missing and should be added so that the reviewers can assess the quality of the western blots presented in this manuscript\n• The legends of the Supplemental Figures is way too brief. Please expand them and describe in more detail what is shown in the figures.\n• Figure S3: I presume these are western blots – against what? GFP?\n• Figure S4: The legend states “Quantitative analysis of EGFP in engineered EVs”. The blot on the left hand site shows “His-EPO standard curve” (what is the connection to EGFP?) and the blot on the right hand site says “Flag”. Again, what is the connection to EGFP? The main text suggests that these blot help in the quantification of EGFP-tagged proteins in EVs, but it is absolutely unclear for me how this is done and how this relates to an anti-FLAG blot and His-tagged EPO.\n• Lines 385-387: The text mentions quantification of EGFP, the corresponding Figure 1e “flag molecules per EV”. Please make this consistent and explain better what has been done.\n• Line 393/394: “Although Rab7a-EVs exhibited the highest EGFP loading, they were internalized less efficiently than ENPP1-EVs and PTGFRN-EVs (Supplementary Figure 5A).” It is unclear to me what is shown in the western blot in Supp. Fig. 5a and how one can see that a) Rab7a-EVs exhibited the highest EGFP loading and b) that they are less efficiently internalized than ENPP1-EVs and PTGFRN-EVs\n• Supplementary Figure 5C: The x-axis is not correct.\n• Lines 399/400: “The truncations and full-length ENPP1 plasmids exhibited comparable levels of EGFP expression in transfected cells (Figure 1H).” This statement is not backed up from the data shown in Fig. 1H, as the statistical test has only been done between untransfected cells and the different expressed proteins. Whether there are differences between the individuals transfected constructs has not been tested.\n• Figure 2A: The GAPDH blot looks strange. Why would GAPDH be present as two different bands in EN144 expressing cells, but only as a single band in untransfected cells?\n• Figure S7: scale bar is not defined\n• Lines 554/555: “Here, we also non-covalent loading of Cas9 protein through selected docking domains, ...” Please correct and rephrase.\n• Lines 604-606: “Genetic fusion of mgp130 or hTNFR1 to the C-terminus of EN144 created constructs, which were transiently expressed in Expi293F cells.” Please explain why murine gp130, but human TNFR1 was used. Further, please state which part of gp130 and TRNFR1 were fused to EN144 (I assume the whole ectodomains without transmembrane and intracellular parts, but this should be stated here).\n• Figure S11B is not explained in the text and should be removed\n• Lines 612-614: “Notably, both EN144-EVmgp130 and EN144-EVhTNFR1 treatments markedly reduced the levels of secreted TNF-α and IL-6 proteins from inflammatory RAW 264.7 cells (Figure 5C-E).” There is no difference between LPS treated and EN144-EVhTNFR1-treated cells in Fig. 5C, and even an increase in the EN144-EVhTNFR1-treated cells in Fig. 5D. The statement is wrong – only ENV133-EV-mgp130 reduces TNF and IL-6 gene expression. Whether any of this treatments has an influence on the protein level is unclear: The quality of the western blots presented in Fig. 5E is too bad to answer this question, and appear to only represent a single experiment. An answer would require better western blots and the quantification of at least three independent experiments.\n• Lines 621/622: “These data prove the anti-inflammatory efficacy of EN144-EVmgp130.” No, they do not. Reduced IL-6 expression level do not prove that something has an anti-inflammatory effect.\n• Lines 637-642: The authors inject tocilizumab into mice and compare this with their EVs. This is highly problematic, because tocilizumab does not work in mice. Tocilizumab binds to the human IL-6R, but not to the mouse IL-6R. See Okazaki et al. Immunol Lett. 2002;84(3):231-40 / Garbers et al. J Biol Chem. 2011;286(50):42959-70 / Nishimoto et al. Ann Rheum Dis. 2000;59 Suppl 1:i21-7 / Ueda et al. Sci Rep. 2013;3:1196 and Lokau et al. PLoS One. 2020 May 4;15(5):e0232612\n• Line 705: The authors evaluate their engineered EVs in a rat model. Again, they use tocilizumab as a control treatment, and again, this is highly problematic, because tocilizumab does also not work in rats. Please see Okazaki et al. Immunol Lett. 2002;84(3):231-40 / George et al. JACC Basic Transl Sci. 2021 Apr 7;6(5):431-443", "reviewer_1_attached": "This manuscript describes a discovery-based project and genetic engineering of cells to endogenously express\nan EV sorting protein/ scaffold. The authors claim that when EVs possess this novel sorting protein, the EVs\nhave enhanced ability to target a cell (when co-expressed with a specific cell targeting protein) and load\ntherapeutic cargo. Based on proteomic studies, the authors purport that a sorting protein, ENPP1, is superior\nto other sorting proteins previously identified, and that a truncated 144 aa ENPP1mversion (EN-144) is even\nmore optimal. The authors then tested the EN-144 expressing EV in several paradigms. The author tests the\nEN-144 in collaboration with gp130, acts as a decoy in IL-6/IL-6 receptor system in models of sepsis and\nosteoarthritis, a degenerative disorder that is highly inflammatory. Cell targeting proteins (CAP) are\nemployed in EVs that target chondrocytes in the osteoarthritis model. A weakness is that the studies to prove\ntargeting are not rigorous. The authors conclude that then EN-144 engineered EVs can target chondrocytes\nand ultimately antagonize inflammation. While the concept of the project is exciting and thoughtful in regard\nto developing a new therapeutic, the manuscript is very dense and lacks sufficient details and controls in\nseveral experiments that dampens this reviewer’s enthusiasm for the work.\nOverall Strengths\n• The authors used 3 distinct methods to isolate EVs which adds to rigor of this EV study and strengthens their\nmass spec results.\n• Discovery approach - performed comprehensive proteomic analyses for sorting proteins that might improve\nthe current standard.\n• Identifying mechanisms that can assist with targeted drug delivery by EV have great therapeutic potential\nis many diseases, not just inflammation\n• In Vivo studies designed to test the safety of the EN-144 EVs\n• Used two distinct models for biological efficacy – sepsis and osteoarthritis Focus on GP130 that has\na broader range than TNFR1\nWeaknesses\n• 1. It is odd that whole cell lysates of EXpi293F do not express CD9 (Fig. 1S) and that CD9 is only observed\nin the EVs.\n• 2. Other cell markers, not just calnexin should be used to confirm the lack of cellular debris. What was the\nviability of the cultured cells prior to exosomes isolation?\n• 3. In Figure 2, the GAPDH immunoblotting is not consistent and difficult to interpret.\n• 4. In Figure 4, the timing of the EV uptake is not clear; was live cell imaging employed? There are no clear methods\nincluded for the EV uptake studies.\n• 5. The Lamp2b-EV high powered image lacks focus compared to the other images. Thus, it is very difficult to\nmake quantitative comparisons.\n• 6. The uptake studies of EV-144-cap are not convincing. Cellular uptake of EVs in 24 hours is a very late timepoint for\nmeasuring EV. The methods for targeting and uptake are not rigorous.\n• 7. Since EVs are heterogenous, the authors results are likely not consistent for all EVs derived from various\nsources.\n• 8. The expression levels of TNFa and IL-6 are not reported in pg/mL or in quantitative units. It’s not clear from the graph\nif the data are fold increase or decrease. The concentration of the cytokines should be revealed.\n• 9. Binding of IL-6 or TNFa to the EN144-EV is not shown. The evidence for being a “decoy” relies on reduced inflammatory mediators in the liver and spleen. Binding kinetics of the EVs to specific inflammatory cytokines,\nlike IL-6, should be shown.\n• 10. Figure 6C shows low magnification images only. Higher magnification should be included."}, "author_response": "We, the authors, are very grateful for your acceptance."}]

Decision letter