Safety Evaluation of Long-term Administration of Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicles

Safety Evaluation of Long-term Administration of Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Safety Evaluation of Long-term Administration of Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicles Ting Lan, Tao-Tao Tang, Jing-Jing Di, Suo-Fu Qin, Aihemaiti Muzaipaier, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6931168/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 17 You are reading this latest preprint version Abstract Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) hold significant therapeutic promise in regenerative medicine; however, their long-term safety profile remains inadequately characterized for clinical translation. In this study, human umbilical cord MSC-derived EVs (hucMSC-EVs) were produced using a heterologous component-free platform by substituting foetal bovine serum (FBS) with human platelet lysate (hPL). A comprehensive safety assessment was conducted by benchmarking hucMSC-EVs against polyethylene glycol (PEG)-liposomes. Pharmacokinetic analysis via cell membrane red fluorescent probe (DID)-labelling revealed rapid clearance of hucMSC-EVs, with a blood half-life of approximately 3 hours and predominant hepatic accumulation. Multi-dose evaluations integrating hematological profiling, cytokines/chemokines quantification, and histopathological scoring demonstrated superior biocompatibility of hucMSC-EVs over PEG-liposomes. Key findings include: 1. hucMSC-EVs did not induce activation of blood inflammatory cells; 2. hucMSC-EVs caused a significant increase in CCL12 only, and the rest of the multiple inflammatory factors/chemokines remained stable; 3. histopathological changes associated with hucMSC-EVs were mainly localized to liver and lungs, yet markedly less severe than PEG-liposome-induced lesions; 4. Transcriptome sequencing further demonstrated that hucMSC-EVs regulated metabolism and complement-coagulation cascade while avoiding activating pro-inflammatory pathways. In contrast, PEG-liposomes up-regulated inflammation-associated pathways such as IL-17 signalling, the MAPK pathway, the TLR pathway, and the T-cell differentiation pathway. This study establishes a multidimensional safety evaluation framework, providing critical preclinical evidence to advance the clinical translation of hucMSC-EVs extracellular vesicles MSC PEG-Liposome safety long-term injection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction MSC-EVs have emerged as transformative nanotherapeutic agents in tissue engineering and regenerative medicine, owing to their unique capacity to deliver bioactive cargo, including nucleic acids, proteins, and lipids, that orchestrate tissue repair through multifaceted mechanisms [ 1 ] . Unlike parental MSCs, MSC-EVs circumvent risks associated with cell transplantation while retaining regenerative potency, positioning them as next-generation acellular therapeutics [ 2 , 3 ] . MSC-EVs exert therapeutic effects across neurological, renal, hepatic, cardiac, and cutaneous disorders through immunomodulation, pro-angiogenic signalling, and direct cell proliferation and differentiation stimulation. [ 4 – 10 ] . Compared to living cell therapies, EVs offer distinct advantages, such as simplified storage, enhanced stability, and ease of handling/administration, thereby significantly facilitating clinical translation [ 11 ] . However, the therapeutic application of MSC-EVs faces critical challenges, particularly concerning biosafety and standardization. Although early-phase clinical trials suggest that MSC-EVs exhibit low acute toxicity [ 12 , 13 ] , their inherent compositional heterogeneity as natural nanoparticles poses unresolved risks. MSC-EVs may carry diverse uncharacterized bioactive components (e.g., donor-specific proteins, non-coding RNAs) capable of triggering unintended immunorecognition or inflammatory cascades [ 14 ] . Such risks are exacerbated in chronic diseases necessitating long-term administration, underscoring the urgent need for systematic safety evaluations. Current safety assessments of MSC-EVs remain fragmented, predominantly focusing on acute toxicity endpoints or isolated immune parameters [ 15 – 17 ] . Given the potential for organ accumulation and delayed immunogenicity, comprehensive characterization of cumulative biosafety risks following repeated MSC-EVs exposure is imperative. To address these gaps, we established a comprehensive safety assessment framework for MSC-EVs through systematic comparative analyses with polyethylene glycol (PEG)-liposomes, which served as a pharmacokinetic-matched controls. This framework enabled multidimensional evaluation of haemodynamics, biodistribution, and long-term safety profiles. We established a xeno-free production platform using human platelet lysate to generate clinical-grade hucMSC-EVs [ 18 ] , eliminating animal-derived contaminants while maintaining EVs functionality. In vivo imaging system (IVIS) imaging revealed pharmacokinetic behavior: hucMSC-EVs exhibited hepatic and splenic tropism and 4-hour circulatory persistence. Our multilayered safety paradigm, including hematological analysis, cytokine storm risk assessment, multi-organ histopathology, and transcriptomics, showed that repeated administration of hucMSC-EVs induced significantly less inflammatory damage and immunogenicity compared to PEG liposomes. These findings validate the translational potential of hucMSC-EV as a low-risk therapeutic platform for regenerative medicine. Results hucMSC-EVs identification and adequate labeling hucMSCs were isolated and expanded under xeno-free conditions using hPL as a serum substitute (Fig. S1A). Flow cytometry analysis confirmed the presence of mesenchymal stromal cell markers, including CD29, CD44, CD90, CD73, and CD105, while showing negative expression of CD45, CD11b, CD34, and HLA-DR in the isolated hucMSCs (Fig. S1B). Furthermore, trilineage differentiation assays validated their multipotency, as evidenced by osteogenic, adipogenic, and chondrogenic differentiation capabilities (Fig. S1C-E). hucMSC-EVs were isolated through sequential ultracentrifugation and size-exclusion chromatography (SEC), and characterized in accordance with the International Society for Extracellular Vesicles (ISEV) 2023 guidelines [19] . Transmission electron microscopy (TEM) imaging revealed the characteristic cup-shaped morphology of hucMSC-EVs (Fig. 1A). Nanoparticle tracking analysis (NTA) demonstrated a median diameter of 146.5 ± 8.3 nm (Fig. 1B). Western blot analysis demonstrated expression of EV-associated markers (Alix, CD9, CD81, Tsg101), while excluding the endoplasmic reticulum (ER) marker Calnexin (Fig. 1C). The results indicated that the positive rate of CD81 in hucMSC-derived EVs was 29.2%. In contrast, the positive rate of CD9 was 20.8% (Fig. 1D). Pharmacokinetics and biodistribution of hucMSC-EVs As natural nanocarriers of bioactive molecules, the circulatory persistence of hucMSC-EVs is critical for optimizing their therapeutic efficacy. To compare the pharmacokinetic behavior of DiD-labeled hucMSC-EVs with PEG-liposomes, we intravenously administered both formulations to 8-week-old C57BL/6 mice and serially monitored blood fluorescence intensity using the IVIS system‌‌. Fluorescence decay curves over time showed that hucMSC-EVs exhibited a blood half-life of approximately 3 h, whereas PEG-liposomes displayed prolonged circulation with a half-life of roughly 6 h. Both agents underwent rapid clearance, with almost complete elimination within 24 h (Fig. 2A). The delayed clearance of PEG-liposomes at 20 hours aligns with prior studies demonstrating that PEGylation enhances hydrophilicity and extends plasma residence time Notably, PEG-liposomes exhibited delayed clearance compared to hucMSC-EVs at 20 hours, consistent with previous reports that PEGylation improves hydrophilicity and prolongs in vivo circulation time [20, 21] . Ex vivo biodistribution profiling demonstrated that hucMSC-EVs accumulated mainly in the liver, and PEG-liposomes mainly in the liver and spleen. (Fig. 2B, C). During the observation period, PEG-liposome accumulation was higher in the spleen than in hucMSC-EVs (Tab. S1, 2). Minimal signals were detected in the non-mononuclear phagocyte system (MPS) organs, including the brain and heart. To corroborate imaging data, tissue sections from the major accumulating organs were collected at 8 hours post-injection. These results are consistent with the overall organ imaging results showing that both PEG-liposomes and hucMSC-EVs were predominantly accumulated in the liver, with the spleen accumulating larger amounts of PEG-liposomes than hucMSC-EVs (Fig. 2D). Notably, fluorescence imaging of the intestine, a representative organ of the digestive tract, revealed negligible signal (Fig. S3A), indicating that EVs and liposomes injected intravenously do not significantly accumulate in the digestive tract. These findings underscore the liver as the primary site of hucMSC-EV accumulation. Multidimensional assessment of the safety of long-term injection of hucMSC-EVs from haematology, serum biochemistry, and cytokine/chemokine analyses. To evaluate the long-term biosafety of hucMSC-EVs, we established a repeated dosing regimen in BALB/c mice: low-dose (10⁹ particles/injection) and high-dose (10¹⁰ particles/injection) groups received intravenous administrations every 3 days for 30 days (10 total injections). In vivo safety was assessed by haematology parameters, serum biochemistry, cytokine/chemokine analyses, histopathology, and RNA-seq after the mid-term (5th injection) and terminal (10th injection) injections (Fig. 3A). Body weight trajectories remained stable across all groups, with variations <5g from baseline (Fig. 3B). Final weight gain analysis revealed comparable growth patterns among cohorts, demonstrating no metabolic disruption induced by hucMSC-EVs (Fig. 3C). Notably, there were no mouse deaths in any of the groups during our observation period. Analysis of complete blood counts after the 5th injection showed that neither low nor high doses of hucMSC-EVs caused a significant elevation of inflammatory cells in the blood. In contrast, PEG-liposomes caused inflammation: low-dose PEG-liposomes(L-Lipo) significantly elevated white blood cell counts (WBC) compared to PBS control group (PBS: 4.93 ±0.81 ×10 9 /L, L-Lipo: 7.13 ± 1.68 ×10 9 /L, high-dose PEG-liposomes(H-Lipo): 5.47 ±1.50 ×10 9 /L, low-dose hucMSC-EVs(L-MSC EV): 3.74±1.47×10 9 /L, high-dose hucMSC-EVs(H-MSC EV): 3.15±0.81×10 9 /L). The counts and proportions of intermediate(Mid) cell and granulocyte (GR) were also elevated in the PEG-liposomes group compared to the PBS control group (Tab. 1). Similarly, after the 10th injection, neither group of hucMSC-EVs caused an elevation of inflammatory cells, whereas persistent WBC activation was observed in the H-Lipo groups(Tab. 2). Critical organ function biomarkers(hepatic enzymes: alanine aminotransferase (ALT) (Fig. 3D) and aspartate aminotransferase (AST) (Fig. 3E), renal parameters: serum creatinine (Scr) (Fig. 3F) and blood urea nitrogen (BUN) (Fig. 3G) remained unaltered across all groups. To investigate potential immune response, we quantified 16 cytokines/chemokines following long-term injections(Fig. 3H, I). CCL27(Fig. 3J), IL-10(Fig. 3K), and CCL22(Fig. 3L) were increased in hucMSC-EVs after five injections compared to the PBS group. Similar increases in both inflammatory factors were observed in the Liposomes group. In addition, CCL22, CCL12, CCL7, IL-6, and CCL11 showed an upward trend in the Liposomes group (all p> 0.05). After ten injections, no significant changes in inflammatory factors were observed in both groups. CXCL5 was up-regulated in the hucMSC-EVs group, and CCL27, IL-10, CCL1, IFN-g, IL-6, CXCL1, CCL17 in the Liposomes group (all p> 0.05). It is worth mentioning that we observed an upregulation of some inflammatory factors in the PBS group during long-term injections as well, which may be due to the endothelial damage of the veins caused by the repeated puncture of multiple injections, which activates platelets and immune cells to release inflammatory factors [22, 23] . In conclusion, our results suggest that long-term administration of hucMSC-EVs caused a lower risk of systemic inflammation compared to PEG-liposomes. Specifically, hucMSC-EV-treated cohorts exhibited stable white blood cell counts and minimal activation of inflammatory cytokine storms, underscoring their superior biocompatibility for chronic therapeutic applications. Histopathology evaluation confirms favorable safety profile of hucMSC-EVs Organ sections collected after 10 intravenous injections were histologically assessed to evaluate potential tissue damage from long-term nanoparticle administration. Both hucMSC-EVs and PEG-liposomes exhibited no significant histopathological alterations in the brain, spleen, or heart, with all scores aligning with the PBS control group (score: 0) (Fig. 4A). This absence of damage may be due to the minimal retention of PEG-liposomes and hucMSC-EVs in these organs, as shown by the organ imaging results mentioned earlier. Hepatocytes in PBS control group displayed characteristic radial plate arrangement around central veins, with intact sinusoidal structures. hucMSC-EV-treated groups showed no significant hepatic damage, with sporadic inflammatory cell infiltration observed in a few mice, without dose-dependent progression. However, the H-Lipo group showed significant inflammatory infiltration compared to the PBS control group ( p< 0.05). (Fig. 4A, B). In the pulmonary pathology, the PBS control group had intact tissue structure with normal alveolar morphology and no inflammatory reaction. The hucMSC-EVs group displayed mild histopathological changes, (L-MSC EV group score:1.29±1.11, H-MSC-EV group score:0.86±1.07), as evidenced by alveolar wall thickening and inflammatory cell infiltration. In contrast, the L-Lipo group showed more severe damage, characterized by inflammatory cell infiltration, alveolar congestion, and alveolar wall thickening (score: 2.6±0.55) (Fig. 4A, C). Control glomeruli exhibited patent capillary loops without proteinaceous deposits. Following 10 injections, L-MSC EV and H-MSC EV groups demonstrated few scattered isolated foci (score: 0.43 ± 0.53), whereas H-Lipo induced significant inflammatory infiltrates (score: 1.2 ± 0.84) (Fig. 4A, D). Collectively, continuous administration of hucMSC-EVs poses a lower risk of tissue and organ lesions compared to PEG-liposomes, affirming their enhanced organ safety profile for long-term therapeutic use. Transcriptomic profiling reveals organ-specific responses to hucMSC-EVs and PEG-Liposomes To elucidate the molecular mechanisms underlying tissue responses, transcriptome sequencing was performed on liver, lung, and kidney tissues from mice receiving 10 injections of hucMSC-EVs or PEG-liposomes. Principal component analysis (PCA) showed strong within-group clustering across all organs (Fig. S4A-C), confirming experimental reproducibility. Differential expression analysis revealed 659 differentially expressed genes (DEGs) (327 upregulated, 332 downregulated) in hucMSC-EV-treated liver tissues, compared to 669 DEGs (492 upregulated, 177 downregulated) in PEG-liposome-treated counterparts. It is noteworthy that 18 inflammation-associated genes (major histocompatibility complex‌ class Ⅱ (H2-Aa), major histocompatibility complex, class II invariant chain (Cd74), myeloperoxidase (Mpo), chemokine (C-C motif) ligand 5(Ccl5), serum amyloid A2(Saa2), etc.) were upregulated in PEG-liposome groups but remained unchanged or downregulated in hucMSC-EV groups (Fig. 5A). KEGG pathway analysis identified distinct enrichment patterns: both groups shared activation in pancreatic secretion, protein and fat digestion and absorption pathways, and MAPK signaling. However, PEG-liposomes uniquely activated immune and inflammatory pathways, including IL-17 signaling, antigen processing and presentation, and T-cell differentiation, whereas hucMSC-EVs modulated homeostatic pathways such as circadian rhythm, cardiac muscle contraction, and endocrine signaling pathways (e.g., cortisol and oxytocin) (Fig. 5B). Differential expression analysis revealed 659 differentially expressed genes (DEGs) (327 upregulated, 332 downregulated) in hucMSC-EV-treated liver tissues, compared to 669 DEGs (492 upregulated, 177 downregulated) in PEG-liposome-treated counterparts. It is noteworthy that 18 inflammation-associated genes (major histocompatibility complex‌ class Ⅱ (H2-Aa), major histocompatibility complex, class II invariant chain (Cd74), myeloperoxidase (Mpo), chemokine (C-C motif) ligand 5(Ccl5), serum amyloid A2(Saa2), etc.) were upregulated in PEG-liposome groups but remained unchanged or downregulated in hucMSC-EV groups (Fig. 5A). KEGG pathway analysis identified distinct enrichment patterns: both groups shared activation in pancreatic secretion, protein and fat digestion and absorption pathways, and MAPK signaling. However, PEG-liposomes uniquely activated immune and inflammatory pathways, including IL-17 signaling, antigen processing and presentation, and T-cell differentiation, whereas hucMSC-EVs modulated homeostatic pathways such as circadian rhythm, cardiac muscle contraction, and endocrine signaling pathways (e.g., cortisol and oxytocin) (Fig. 5B). The kidneys showed similar between-group differences to the lungs. There are 713 DEGs (308 upregulated, 405 downregulated) in hucMSC-EVs-treated kidneys, and 539 DEGs (247 upregulated, 292 downregulated) in PEG-liposomes-treated kidneys. Like the hepatic response pattern, hucMSC-EVs predominantly influenced steroidogenesis and lipid metabolism, while PEG-liposomes sustained the activation of immune pathways. In addition, hucMSC-EV-treated kidneys were consistently enriched in complement components, with lungs showing differential expression of 11 complement-related genes, such as complement component 6(C6), complement component 9(C9), alpha2-HS glycoprotein (Ahsg), and vitronectin (Vtn), in hucMSC-EV and PEG liposomes. hucMSC-EVs consistently upregulated complement and coagulation cascades in lung and kidney tissues (Fig. 5E, F). This coordinated regulation of interconnected homeostatic systems suggests a potential immunomodulatory mechanism for hucMSC-EVs biocompatibility. Consistent with hematological and histopathological findings, transcriptomic signatures indicate that long-term hucMSC-EVs injections elicit significantly lower inflammatory pathway activation than PEG-liposomes across multiple organ systems. Discussion The core bottlenecks in the current clinical application of MSC-EV are fluctuations in therapeutic efficacy due to heterogeneous sources, insufficient target delivery efficiency, and, more importantly, the lack of comprehensive safety assessment and quality control standards to ensure therapeutic efficacy and avoidance of side effects and to promote the development of clinical applications. Our study evaluated the safety of hucMSC-EVs in terms of biodistribution, blood cytology assay, serum biochemistry assay, serum inflammatory factors and chemokines assay, pathological damage score, and RNA-seq. It demonstrated that it does not cause significant inflammatory response and tissue damage and has a good safety profile for long-term administration. We injected mice with DiD-labelled hucMSC-EVs and PEG-liposomes, using serum and organ imaging acquired at different time points after injection. We found that hucMSC-EVs were metabolised slightly faster than PEG-liposomes, but the metabolic trends of both were highly similar. This parallelism suggests that despite the diverse biological origins and molecular compositions of nanoscale carriers, whether endogenous EVs or synthetic liposomes, their systemic fates may be governed by common biophysical principles. Whereas, existing studies have clarified that the blood residence time and organ targeting efficiency of nanoparticles are mainly regulated by the following three physical factors, including EV size, surface charge, and membrane fluidity [ 24 ] . Among these, nanoparticle size is the major influencing factor, affecting their uptake, biodistribution, and circulating half-life. The bEVs (> 200 nm) and sEVs (< 200 nm) of invasive triple-negative breast cancers predominantly target the lungs, liver, and spleen (sEV only) at low doses, and sEVs are more readily transported from other major organs to the liver over time [ 25 ] . Smaller-sized EVs can more easily penetrate the vascular endothelial barrier and have unique biodistribution properties [ 26 , 27 ] . In addition, during culture and after entering the somatic circulation, EVs are exposed to various proteins that bind to the EVs to form protein crowns, which in turn affect the behaviour of the EVs [ 28 , 29 ] . It was found that pre-coating EVs with albumin to create albumin-rich protein corona-EV complexes enhanced non-phagocytic uptake in the liver [ 30 ] , a phenomenon that could be used as a camouflage strategy to enhance EV targeting. Beyond these inherent properties, emerging engineering strategies can further reprogram EV biodistribution. Targeted delivery of EVs can also be facilitated by altering the in vivo distribution of EVs by adding homing peptides to the EV surface. For example, peptides such as RGD and its cyclic variant c(RGDyK) bind target cells [ 31 , 32 ] , while cardiomyocyte-specific peptides (CMP) guide EVs to heart tissue [ 33 ] . Other reported sequences include ASSLNIA and CSTSMLKAC (targeting muscle and ischemic myocardium), a series of T140 variants (T140, Scr‑T140, T140‑KLA, B‑TL5) directed at CXCR4‐expressing cells [ 34 ] , PTHTRWA for lung cancer targeting, an EGFR‑targeting peptide [ 35 ] , CKBP for aneurysmal smooth muscle cells, and a Lys‑Leu‑Ala peptide used with low‑density lipoprotein mimetics for glioma [ 36 , 37 ] . In addition, modification of extracellular vesicle glycosylation can alter their in vivo distribution. The investigators treated EVs from mouse liver proliferating cells with neuraminidase to digest the glycoprotein-terminal sialic acid residues on the surface of the EVs. They found that the modified EVs induced accumulation in the lungs when injected intravenously, and additionally, when injected through the tarsal joints, the neuraminidase-treated vesicles were better distributed in the axillary lymph nodes than the untreated EVs [ 38 ] . The results of our multidimensional safety assessment based on MSC-EVs showed a clear safety profile of MSC-EVs.The superior safety profile of MSC-EVs compared to MSCs is since they do not express major histocompatibility complex (MHC) class I/II antigens on their surfaces, which considerably reduces the risk of immune rejection and tumour formation‌ [ 39 ] . In vitro toxicological studies have demonstrated that MSC-EVs are safe, with no detectable genotoxic effects, haemolysis, or significant effects on platelet aggregation [ 15 ] . Available clinical trials have shown that MSC-EVs are safe for various diseases with few adverse effects [ 40 – 42 ] . However, existing safety evaluations remain fragmented, often neglecting dose-dependent responses, immunogenicity, and mechanistic insights. The basic factors affecting nanoliposome safety include composition, size, surface charge, stability, release of dopant drug, and penetration into tissues [ 43 ] . The advantage of cationic surface liposomes is that they can bind negatively charged cargoes through electrostatic interactions. Although unmodified cationic liposomes have no or minimal toxicity at low doses, some cytotoxic effects have been reported. For example, cationic liposomes are toxic when administered in multiple doses, whereas neutral liposomes are relatively safe. Studies have shown no change in mortality in mice in the long-term treatment group with neutral liposomes. In contrast, the mortality rate of mice in the long-term treatment group with positively charged liposomes was 45 percent [ 44 ] . Furthermore, positively charged liposomes lead to more severe changes in blood levels and inflammatory components than neutral liposomes. This may be due to surface positively charged groups inducing ROS production in cells [ 45 ] and cationic liposomes interacting with the plasma membrane of neutrophils and triggering neutrophil stimulation [ 46 ] . Polyethylene glycolisation effectively prolongs the systemic circulation time of LNPs, thereby greatly improving their pharmacokinetics and efficiency [ 47 ] . However, the novel PEG liposomes are not completely non-toxic and may present a risk of cytotoxic tissue oxidative damage [ 46 , 47 ] . Repeated injections of PEG liposomes may cause an unexpected immunogenic response, producing antibodies to PEG and leading to accelerated blood clearance (ABC) [ 48 – 50 ] . In addition, side effects such as acute infusion reactions and hand-foot syndrome (HFS) induced by liposomal drugs cannot be ignored, as documented by clinical studies [ 51 ] . Our finding revealed that long-term injection of PEG liposomes did not cause death in mice, but an increase in blood leukocyte counts was observed in the L-Lipo group and the H-Lipo group after 5 sustained injections, and in the H-Lipo group after 10 injections, which is consistent with other literature reporting that liposomes may cause blood inflammation. Pathological damage in long-term injections of PEG-Liposomes was mainly concentrated in the liver and lungs. Still, the pathological damage scores were less than or equal to 3 for all organs (the most severe damage was 4). In addition, long-term injection of PEG-Liposomes caused activation of hepatic, pulmonary, and renal IL-17 signalling, T-cell differentiation, TLR pathway, and other immune and inflammation-related pathways in mice. Our comprehensive safety evaluation revealed that hucMSC-EVs have a good safety profile even after long-term injections, with three significant findings: 1. hucMSC-EVs have a faster blood clearance rate than PEG-liposomes, and their main organ of distribution in the body is the liver; 2. hucMSC-EVs are highly biocompatible, and long-term injections cause less immune activation; 3. hucMSC-EVs transcriptome features show an enrichment of metabolic homeostasis rather than inflammatory responses, in addition to the unique complement-coagulation cascade regulation of hucMSC-EVs. hucMSC-EVs have better biocompatibility and less immune activation caused by long-term injection; 3. hucMSC-EVs transcriptome features show enrichment in metabolic homeostasis rather than inflammatory response, and in addition, the unique complement-coagulation cascade regulation of hucMSC-EVs may be responsible for the immune tolerance mediated by hucMSC-EVs. Comprehensive safety assessment of MSC-EVs is not only about individual therapeutic risk control, but also a core aspect of translation from basic research to clinical application. In conclusion, our study demonstrated the safety of long-term administration of hucMSC-EVs, and laid a sure foundation for the future development of MSC-EVs as drug delivery vehicles and clinical translation. Declarations Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (82230022, 82030024, 81720108007), the National Key Research and Development Program of China (2022YFC2502500), the Joint Innovation Laboratory for the Industrialization of Extracellular Vesicle-Based Drug Delivery Technology Established by Shenzhen Kexing Pharmaceutical Co., Ltd. and Southeast University (8524006050) to B.-C.L. This research was supported by additional grants from National Natural Science Foundation of China (82200772), the Natural Science Foundation of Jiangsu Province grant (BK20220828), the Fundamental Research Funds for the Central Universities (2242023R40030), and the Start-up Research Fund of Southeast University (RF1028623235) to T.-T.T. It is also supported by Shenzhen Major Science and Technology Project (KJZD20230923115201003) and Beijing Echo Biotech Co., Ltd. Methods hucMSC Extraction and culture All umbilical cord donors provided their informed consent. All procedures were approved by the Ethical Committee for Clinical Research of Zhongda Hospital, Affiliated to Southeast University (Approval No. 2023ZDSYLL118-P01). After rinsing the umbilical cord twice with pre-cooled sterile PBS, the amniotic membrane, two arteries, and one vein were removed with forceps, and the remaining umbilical cord tissue was cut into pieces of 1-3 mm 3 with a piece of Warton's glue. The pieces of umbilical cord tissue were transferred into a 50 mL centrifuge tube, 20 mL of PBS was added, and centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was discarded, and the pre-configured complete medium(base medium(MSC NutriStem® XF Medium, 05-200-1A, Sartorius)containing 5% hPL (PLTGOLD100R, Sartorius) )for hucMSCs was added, and the medium containing the pieces of umbilical cord tissue was transferred into a T75cm 2 cell culture flask. Cultivate the cells at 37℃ in a 5% CO 2 incubator, and change the medium half every three days, and cells can be seen crawling out around the tissue mass in about one to two weeks. When the cell fusion around the tissue block reaches 80%-85%, the cells can be passed on. After the cells were washed with sterile PBS once, add 3ml TrypleLE Express (12604039, Gibico), digest the cells for about 2 min, add an equal amount of fresh medium to neutralize, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh complete medium and mix gently by blowing, and then transfer to new culture flasks for passaging culture. hucMSC-EVs Isolation, Purification, and Storage When the hucMSCs grew to 75%-80% fusion, discard the old medium and wash the cells twice with PBS; add the basal medium DMEM/F12(Gibico, 11320033) and incubate the cells in a 37°C, 5% CO2 incubator for 48 h. Collect the supernatant into a centrifuge tube and centrifuge the cells at 4 ℃ for 20 min at 2,000 g. Transfer the supernatant into another centrifuge tube and centrifuge the supernatant at 4 ℃ and 13,500 g for 30 min. The supernatant was transferred to another centrifuge tube and centrifuged at 4 ℃ and 13500 g for 30 min; the supernatant was taken into an ultracentrifuge tube and ultracentrifuged at 4 ℃ and 100000 g for 2 h. The precipitates were resuspended by adding an appropriate amount of sterile PBS filtered through a 0.22 μm filter to obtain the preliminary extracted hucMSC-EVs. The size exclusion chromatography (SEC) Exo exclusion column(Echo9101A-S10, Echo Biotech) was equilibrated to room temperature, 20 mL of PBS was used to rinse the column, 1 mL of preliminary extracted hucMSC-EVs was added, and when all of the sample was in the separation column, PBS was added for elution, 500 ul for 1 fraction, and the EV-enriched fractions 3-7 were collected after purification. hucMSC-EVs were freshly extracted and stored at −80 °C for no more than one month before usage. hucMSC-EVs Detection Surface marker profiling was performed through immunoblotting analysis. Briefly, exosome-specific proteins (Alix [ab117600, Abcam], CD9 [ab236630, Abcam], CD81 [ab97559, Abcam], Tsg101 [ab125011, Abcam]) and the endoplasmic reticulum marker Calnexin (ab22595, Abcam) were probed using Western blot. For nanoparticle characterization, EV samples were subjected to ExoView™ platform (NanoView Biosciences, Boston, MA) analysis following the manufacturer's protocol. Prior to immunofluorescence staining, EV concentrations quantified via nanoparticle tracking analysis (NTA) were adjusted to 1–3 × 10⁸ particles/mL by 1:1 dilution with incubation buffer. Nano flow cytometry Load 30ul hucMSC(10 10 ) into EP tube for each component, add 1 test amount of antibody(CD9,BD Pharminge,555371) (CD81,BD Pharminge,551108) according to the antibody instruction, mix thoroughly, and incubate for 1 hour at 37℃, protected from light. After incubation, add 1ml of 0.22 um filtered PBS to transfer the EV to the ultra-isolated tube, and then ultracentrifuge at 4℃, 100000g for 75min after leveling. Avoid light as much as possible and resuspend the EV with 30-50 ul PBS in a 600 ul EP tube. Power on the nanofluidizer and start the system, after completing the routine maintenance and quality control, put the stained samples on the sample stage, turn on the switch of the fluorescence channel that the sample is to be detected, and then the software will automatically set the threshold value, collect and save the sample data. Fluorescent labeling and free dye purification of hucMSC-EVs and Liposomes We labeled hucMSC-derived extracellular vesicles (EVs) and PEG-liposomes with 1 mM of DiD dye (C1039, Beyotime) and incubated them at room temperature in the dark with a shaking speed of 40 rpm for 2 hours. Subsequently, unbound free dye was removed using size-exclusion chromatography (SEC) as previously described. Preliminary experiments involved collecting fractions 1-15, where we assessed the fluorescence intensity of different fractions using a multifunctional microplate reader (Fig. S2A). Additionally, electron microscopy analysis (Fig. S2D) and Western blot (WB) detection of the EV markers were performed to confirm that EVs were predominantly enriched in fractions 4-7. Consequently, we pooled fractions 4-7 for further analysis. Finally, we conducted a preliminary evaluation of the fluorescence positivity rate of the labeled liposomes and hucMSC-EVs using nanoparticle tracking analysis to assess the impact of DiD labeling (Fig. S2E). Blood metabolism and distribution imaging after MSC-EVs or PEG-liposomes injection Eight-week-old C57BL/6J mice (purchased for the experiments from: Beijing Vital River Laboratory Animal Technology Co., Ltd., China) weighing 20-23 g were selected and fasted for 12 h. Mice were injected into the tail vein with 200 uL of 1×1010 particles DiD-labeled MSC-EVs, PEG-liposomes, or PBS. Mice were anaesthetised by intraperitoneal injection of tribromoethanol at a concentration of 1.25% at a dose of 200 ul per 10 g of mouse body weight, and the blood was collected from the heart into a 1.5 mL EP tube and left to stand at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min, After injection, mice were executed at different time points: 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h, and blood was collected from the heart into 1.5mL EP tubes, which were left at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min, and the upper layer of the serum was removed, and the brain, heart, liver, lungs, spleen, kidneys, and gastrointestinal tract were retained, and the serum and the organisms were subjected to optical imaging, respectively. Fluorescence values were normalized to the 0.5-hour baseline measurement to calculate relative blood retention percentages. Routine blood tests and biochemistry testing of blood samples Blood samples for routine blood tests were added to 1.5 mL EP tubes pre-filled with anticoagulant(YA1461, Solarbio). The automatic blood cell analyzer( DF62 lab, DYMIND) starts automatic measurement after setting the species. Determination of serum cytokines by liquid-phase suspension microarray technology Microbeads were shaken with a shaker, at 1,400 rpm, for 30 seconds and then 50 ul per well was added to the 96-well plate. After 4-fold dilution of each sample, take 50ul of the sample, the standard and the blank control were prepared according to the instructions, also take 50ul of the sample, affixed with a sealing film, and placed on a plate shaker at 850 rpm, protected from light, at room temperature, and incubated for 30 min. Discard the sample, use a plate washer to wash it 3 times, and then dilute it using the Antibody Diluent according to the instructions. DetectionAntibody, 25 μL of diluted DetectionAntibody was added to each well, sealed with a sealing film, and incubated on a plate shaker with shaking at 850 rpm, protected from light, and at room temperature for 30 min. Samples were discarded, washed 3 times with a plate washer, and diluted with Assay Buffer as described in the instructions. Streptavidin-PE, add 50 μL of diluted Streptavidin-PE to each well, affix a sealing film, and incubate for 10 min at 850 rpm on a plate shaker with shaking, protected from light, and at room temperature. Wash three times with a plate washer, and then resuspend with 125 μL of Assay Buffer to each well, affix a sealing film, and incubate for 30 min at 850 rpm, room temperature, and protected from light. 850 rpm, room temperature, protected from light, shaken for 30 s and placed in a calibrated Bio Plex 200 machine to read the values. Hematoxylin-Eosin staining HE staining was performed using 3μm thick paraffin-embedded tissue sections. Tissue sections were placed in a thermostat box and baked at 60°C for 1 h. Sections were deparaffinized twice in a xylene vat for 15 min each time, then sequentially placed in a gradient ethanol vat for hydration, and the sections were rinsed in ddH2O for 5 min. Sections were placed in Hematoxylin Staining Solution for 10 min, slowly rinsed in ddH 2 O for 10 min, decolorized in hydrochloric acid-alcohol for 20 s, and rinsed in ddH 2 O for 10 min, and returned to blue in ammonia water for 3 min. After returning to blue for 3 min, ddH 2 O was rinsed for 10 min, dehydrated in 70% and 90% alcohol for 10 min each, staining with eosin staining solution for 2-3 min, dehydration in gradient ethanol, followed by xylene transparency for 5 min, air drying, and sealing with drops of neutral gum and coverslips. Histopathological damage score Systematic histomorphological evaluation was performed on paraffin-embedded sections of six major organs following 5 and 10 times injections. Tissue injury was quantified using a 5-tier histopathological scoring system (0: intact microstructure; 1 : slight damage (occasional cellular degeneration or inflammation); 2 : mild damage (localized cellular degeneration or mild inflammation); 3 : moderate damage (marked cellular degeneration or moderate inflammatory cell infiltration) 4: severe necrosis/architectural disruption (extensive cellular necrosis or severe disruption of tissue structure)). Transcriptome sequencing Total RNA was extracted with TRIzol, and then the purity and concentration of RNA were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Transcriptome libraries were constructed using the VAHTS Universal V5 RNA-seq Library Prep Kit and sequenced using the llumina Novaseq 6000 sequencing platform. Reference genome comparisons were performed using HISAT2 software, gene expression (FPKM) calculations were performed, and read counts (counts) for each gene were obtained by HTSeq-count. PCA analysis of genes (counts) as well as mapping was performed using R (v 3.2.0) to assess sample biological replicates. Differentially expressed genes were analyzed using DESeq2 software, where genes that met the thresholds of q-value 2 or foldchange < 0.5 were defined as differentially expressed genes (DEGs). Hierarchical clustering analysis of DEGs was performed using R (v 3.2.0) to demonstrate the expression patterns of genes across groups and samples. Radar plots were created for the top 30 genes using the R package ggradar to demonstrate changes in expression of up- or down-regulated genes. Subsequently, GO enrichment analysis of differentially expressed genes based on the hypergeometric distribution algorithm was used to screen for significantly enriched functional entries. R (v 3.2.0) was used to plot bar charts, chord charts or enrichment analysis circle plots for significantly enriched functional entries. Statistical analysis Data were expressed as mean ±SD. Statistical analysis was performed using a twotailed Student’s t test or one-way analysis of variance (ANOVA), with P < 0.05 considered significant. References JEPPESEN D K, FENIX A M, FRANKLIN JL et al. Reassessment Exosome Composition [J] Cell, 2019, 177(2). RANI S, RYAN A E, GRIFFIN M D, et al. Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications [J]. Mol Ther. 2015;23(5):812–23. ZHANG K, CHENG K. 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Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo [J]. Adv Mater. 2023;35(13):e2208966. ABRA R M, HUNT CA. Liposome disposition in vivo. III. Dose and vesicle-size effects [J]. Biochim Biophys Acta. 1981;666(3):493–503. SOO C Y, SONG Y, ZHENG Y, et al. Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells [J]. Immunology. 2012;136(2):192–7. XIAO Q, ZOULIKHA M. The effects of protein corona on in vivo fate of nanocarriers [J]. Adv Drug Deliv Rev. 2022;186:114356. HEIDARZADEH M, ZAREBKOHAN A, RAHBARGHAZI R, et al. Protein corona and exosomes: new challenges and prospects [J]. Cell Commun Signal. 2023;21(1):64. LIAM-OR R, FARUQU F N WALTERSA, et al. Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent [J]. Nat Nanotechnol. 2024;19(6):846–55. SHEIKH A, MD S. RGD engineered dendrimer nanotherapeutic as an emerging targeted approach in cancer therapy [J]. J Control Release. 2021;340:221–42. AL FARUQUE H, CHOI E-S, KIM J-H, et al. Enhanced effect of autologous EVs delivering paclitaxel in pancreatic cancer [J]. J Control Release. 2022;347:330–46. MENTKOWSKI K I, TARVIRDIZADEH T, MANZANERO C A, et al. Surface engineering enhances the therapeutic potential of systemically delivered extracellular vesicles following acute myocardial infarction [J]. FASEB J. 2024;38(18):e70070. JAYASINGHE M K, PIRISINU M, YANG Y, et al. Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy [J]. Theranostics. 2022;12(7):3288–315. PHAM T C, JAYASINGHE M K, PHAM T T, et al. Covalent conjugation of extracellular vesicles with peptides and nanobodies for targeted therapeutic delivery [J]. J Extracell Vesicles. 2021;10(4):e12057. ANTES T J, MIDDLETON R C, LUTHER K M, et al. Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display [J]. J Nanobiotechnol. 2018;16(1):61. YE Z, ZHANG T, HE W, et al. Methotrexate-Loaded Extracellular Vesicles Functionalized with Therapeutic and Targeted Peptides for the Treatment of Glioblastoma Multiforme [J]. ACS Appl Mater Interfaces. 2018;10(15):12341–50. ROYO F, COSSíO U, RUIZ DE ANGULO A, et al. Modification of the glycosylation of extracellular vesicles alters their biodistribution in mice [J]. Nanoscale. 2019;11(4):1531–7. REZAIE J, NEJATI V, MAHMOODI M, et al. Mesenchymal stem cells derived extracellular vesicles: A promising nanomedicine for drug delivery system [J]. Biochem Pharmacol. 2022;203:115167. GIOVANNELLI L, BARI E, JOMMI C, et al. Mesenchymal stem cell secretome and extracellular vesicles for neurodegenerative diseases: Risk-benefit profile and next steps for the market access [J]. Bioact Mater. 2023;29:16–35. DOEPPNER T R, HERZ J, GöRGENS A, et al. Stem Cells Transl Med. 2015;4(10):1131–43. Extracellular Vesicles Improve Post-Stroke Neuroregeneration and Prevent Postischemic Immunosuppression [J]. SHI M-M, YANG Q-Y MONSELA, et al. Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles [J]. J Extracell Vesicles. 2021;10(10):e12134. TERESHKINA Y A, TORKHOVSKAYA T I, TIKHONOVA E G, et al. Nanoliposomes as drug delivery systems: safety concerns [J]. J Drug Target. 2022;30(3):313–25. HE K, TANG M. Safety of novel liposomal drugs for cancer treatment: Advances and prospects [J]. Chem Biol Interact. 2018;295:13–9. KNUDSEN K B, NORTHEVED H, KUMAR P E K, et al. In vivo toxicity of cationic micelles and liposomes [J]. Nanomedicine. 2015;11(2):467–77. HWANG T-L, HSU C-Y, ALJUFFALI I A, et al. Cationic liposomes evoke proinflammatory mediator release and neutrophil extracellular traps (NETs) toward human neutrophils [J]. 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Meta-analysis of clinical and preclinical studies comparing the anticancer efficacy of liposomal versus conventional non-liposomal doxorubicin [J]. J Control Release. 2016;232:255–64. Additional Declarations No competing interests reported. Supplementary Files WBuncroppedimages.pdf floatimage1.jpeg Figure abstract Tables.docx Supp.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 08 Jul, 2025 Reviewers agreed at journal 05 Jul, 2025 Reviewers agreed at journal 05 Jul, 2025 Reviews received at journal 04 Jul, 2025 Reviewers agreed at journal 04 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 23 Jun, 2025 Submission checks completed at journal 23 Jun, 2025 First submitted to journal 19 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6931168","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476425333,"identity":"b425adc4-beec-4468-ab2b-c6124c8d3fcd","order_by":0,"name":"Ting Lan","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Lan","suffix":""},{"id":476425334,"identity":"222875c3-cedb-4648-a6c6-0a6624074fa0","order_by":1,"name":"Tao-Tao Tang","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Tao-Tao","middleName":"","lastName":"Tang","suffix":""},{"id":476425335,"identity":"572f7c82-4b72-4713-8956-a293442bd6ac","order_by":2,"name":"Jing-Jing Di","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Jing-Jing","middleName":"","lastName":"Di","suffix":""},{"id":476425336,"identity":"c4cba808-100a-4858-b065-5c3ec5ed7751","order_by":3,"name":"Suo-Fu Qin","email":"","orcid":"","institution":"Shenzhen Kexing Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Suo-Fu","middleName":"","lastName":"Qin","suffix":""},{"id":476425337,"identity":"fbf7bcd7-0368-4c54-bded-d751c00b440b","order_by":4,"name":"Aihemaiti Muzaipaier","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Aihemaiti","middleName":"","lastName":"Muzaipaier","suffix":""},{"id":476425338,"identity":"71c5bf57-2c25-4b86-8d2f-1c504e2261ff","order_by":5,"name":"Qin-Yu Zhou","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Qin-Yu","middleName":"","lastName":"Zhou","suffix":""},{"id":476425339,"identity":"98392107-b979-494e-8b5d-9c77e69efbda","order_by":6,"name":"Bi-Cheng Liu","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Bi-Cheng","middleName":"","lastName":"Liu","suffix":""},{"id":476425340,"identity":"f760d7d5-4fdf-4039-a74a-f708f86ade05","order_by":7,"name":"Qiu-Li Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDACdiD+AGEaEKmFmYGBcQbJWph5SNLCd5jHTNrmj01iA3vzNgmGmjuEtUiCtOS2pSU28Bwrk2A49oywFoPDvNukcxsOJzZI5JhJMDYcJlKLxZ//iQ3yb0jRwsB2AGgLD5FaJA/zf7bsbUs2buNJK7ZIOEaEFr7jbYk3fvyxk+1nP7zxxocaIrQwHIDSbCAigQgNCC2jYBSMglEwCnACAAFeNTZC6U/MAAAAAElFTkSuQmCC","orcid":"","institution":"Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Qiu-Li","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-06-19 12:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6931168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6931168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85562504,"identity":"41fd6702-ea98-4a51-bbd9-4d736312af0b","added_by":"auto","created_at":"2025-06-27 13:32:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":139630,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of hucMSC-EVs. (A) TEM images of MSC-EVs. Scale bars show 500 and 100 nm. (B) NTA detection of hucMSC-EVs median particle size: 146.5 nm. (C) Western blot identification of EV markers: Alix, CD9, CD81, TSG101, and ER marker: Calnexin. (D, E) EV markers on hucMSC-EVs were detected by nanoflow cytometry. The positivity rate was 29.2% for CD9(D) and 20.8% for CD81(E).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/ff6199d9865fe78b272fde48.jpg"},{"id":85562506,"identity":"cb2e99ce-140f-4b65-95aa-d5c528dca8c0","added_by":"auto","created_at":"2025-06-27 13:32:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":474106,"visible":true,"origin":"","legend":"\u003cp\u003eBlood metabolism and organ distribution of hucMSC-EVs and PEG-liposome in vivo. (A) Percentage fluorescence decay in mouse blood at various time points after injection, n=5. (B, C) Fluorescence distribution imaging and fluorescence intensity analysis of each organ in the DiD-Labeled hucMSC-EVs group(B) and DiD-Labeled PEG-Liposomes group(C). (D) Fluorescence confocal images of liver, lung, spleen, and kidney tissue sections following injection of DiD-labeled PEG-liposomes and DiD-labeled hucMSC-EVs, blue represents DAPI, and red represents DiD staining. Scale bars show 20 μm.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/56dc1414eb1f856a040e693d.jpg"},{"id":85563176,"identity":"45224813-5514-4990-be4f-db31a4e6e8e4","added_by":"auto","created_at":"2025-06-27 13:40:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333596,"visible":true,"origin":"","legend":"\u003cp\u003eBody Weight, Liver and Kidney Function, and Inflammatory Factors in Mice Chronically Injected with PEG-liposomes and hucMSC-EVs. (A) Schematic diagram of the experimental design. Briefly, mice were injected with PBS, L-MSC EV, H-MSC EV, L-Lipo, and H-Lipo every 3 days, and blood samples and organ samples were taken after 5 and 10 injections for relevant experiments, including hematology， serum biochemistry， cytokine/chemokine analyses， histopathology， and RNA-seq. Created using BioRender.com. (B) Body weight change curve of mice injected with hucMSC-EVs and PEG-liposomes over a specified period. n=5. (C) The ratio of body weight to initial body weight in mice after 10 injections of hucMSC-EVs and PEG-liposomes was expressed as a percentage. n=5. (D, E) The serum levels of ALT (D) and AST (E) in each group. n = 4. (F, G) The serum levels of BUN (F) and Scr (G) in each group. n = 5. (H, I) Heatmap of serum cytokine/ chemokine profiles in each group after 5 injections(H) and 10 injections(I), including BCA-1/CXCL13, CTACK/CCL27, ENA-78/CXCL5, Eotaxin-2/CCL24, Eotaxin/CCL11, IL-10, CCL1, IFN-g, IL-6, KC/CXCL1, MCP-3/CCL7, MCP-5/CCL12, MDC/CCL22, MIP-1a/CCL3, TRAC/CCL17, TNF-a. n=5. (J-L) The serum concentrations of CCL27 (J), IL-10 (K), and CCL22 (L) in each group after 5 \u0026nbsp;injections. n = 5.*\u003cem\u003ep\u0026lt;\u003c/em\u003e 0.05, *** \u003cem\u003ep\u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/4f93b752236f46090d3cc03c.jpg"},{"id":85562509,"identity":"8db9602e-b831-47da-9c68-879eadb46f3f","added_by":"auto","created_at":"2025-06-27 13:32:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1182173,"visible":true,"origin":"","legend":"\u003cp\u003eAfter 10 injections, representative microphotographs and histopathological scoring of brain, heart, liver, spleen, lung, and kidney tissues were obtained. (A) H\u0026amp;E staining of major organs, including brain, lung, heart, liver, spleen, and kidney tissues from mice in each group. Scale bars show 100 μm. The red arrows in the figure point to inflammatory cell infiltration, and the blue arrows point to blood congestion. \u0026nbsp;(B-D) Stacked bar chart showing the distribution of histopathological scores (1-4) in liver \u0026nbsp;(B), lung (C), and kidney (D) tissues after 10 injections, n=8. *\u003cem\u003ep\u0026lt;\u003c/em\u003e 0.05, **\u003cem\u003ep\u0026lt;\u003c/em\u003e 0.01, ****\u003cem\u003ep\u0026lt;\u003c/em\u003e 0.0001 vs PBS group.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/f5c967d7f3042b8fe1f3c19c.jpg"},{"id":85563177,"identity":"95cb1b38-e5f1-4cfc-8043-c5ac76c45c38","added_by":"auto","created_at":"2025-06-27 13:40:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":410036,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic sequencing of liver, lung, and kidney tissues. (A, C, E) Volcano Plot of DEGs in liver tissue(A), lung tissue(C), and kidney tissue(E) from the hucMSC-EV and PEG-Liposome groups, intersecting clusters of inflammatory genes in both groups. The X-axis represents the logarithmic fold change in gene expression (Log2 Fold Change), while the Y-axis indicates gene significance (-Log10(p-value)). Red points signify upregulated genes (\u003cem\u003ep\u0026lt;\u003c/em\u003e 0.05 and |Log2 Fold Change| \u0026gt; 1), whereas blue points denote downregulated genes. The significance threshold is set at \u003cem\u003ep\u0026lt;\u003c/em\u003e 0.05. (B, D, F) Liver(B), lung(D), and kidney-specific(F) differentially expressed genes were analyzed for KEGG pathway enrichment to identify the top 10 pathways. The resulting graphs were partitioned into two sides: on the left, pathways enriched in PEG-liposomes treatment, and on the right, pathways enriched in hucMSC-EVs treatment. Larger bubbles indicate more differentially expressed protein-coding genes, with colors ranging from blue to red based on significance (lower p-values).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/ad618a320712851303532865.jpg"},{"id":85564099,"identity":"e621003b-9e7c-4c08-99e4-45e8ff0f858b","added_by":"auto","created_at":"2025-06-27 13:56:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3366669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/1366df43-ca86-4864-8fe2-b3798945ba75.pdf"},{"id":85563191,"identity":"6ebf6de9-ef0d-4cda-b1f5-1813f787304e","added_by":"auto","created_at":"2025-06-27 13:40:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13226038,"visible":true,"origin":"","legend":"","description":"","filename":"WBuncroppedimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/8065266f79e63c704825bf2c.pdf"},{"id":85562513,"identity":"be613e67-2f84-4499-947b-f64b15c63abe","added_by":"auto","created_at":"2025-06-27 13:32:17","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":373406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/2b6d7e142701c36319dfd5e2.jpeg"},{"id":85562512,"identity":"4901a406-4081-407b-aeb2-2a1b6bad0fbb","added_by":"auto","created_at":"2025-06-27 13:32:17","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":412346,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/38be9692ef7c638cc7bd5196.docx"},{"id":85562514,"identity":"6c3cf0f7-c698-44f3-baff-6f55eade2068","added_by":"auto","created_at":"2025-06-27 13:32:18","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1964820,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-6931168/v1/9782a9751ff78e5b665f864d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Safety Evaluation of Long-term Administration of Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMSC-EVs have emerged as transformative nanotherapeutic agents in tissue engineering and regenerative medicine, owing to their unique capacity to deliver bioactive cargo, including nucleic acids, proteins, and lipids, that orchestrate tissue repair through multifaceted mechanisms\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Unlike parental MSCs, MSC-EVs circumvent risks associated with cell transplantation while retaining regenerative potency, positioning them as next-generation acellular therapeutics\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. MSC-EVs exert therapeutic effects across neurological, renal, hepatic, cardiac, and cutaneous disorders through immunomodulation, pro-angiogenic signalling, and direct cell proliferation and differentiation stimulation.\u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCompared to living cell therapies, EVs offer distinct advantages, such as simplified storage, enhanced stability, and ease of handling/administration, thereby significantly facilitating clinical translation\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the therapeutic application of MSC-EVs faces critical challenges, particularly concerning biosafety and standardization. Although early-phase clinical trials suggest that MSC-EVs exhibit low acute toxicity\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, their inherent compositional heterogeneity as natural nanoparticles poses unresolved risks. MSC-EVs may carry diverse uncharacterized bioactive components (e.g., donor-specific proteins, non-coding RNAs) capable of triggering unintended immunorecognition or inflammatory cascades\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Such risks are exacerbated in chronic diseases necessitating long-term administration, underscoring the urgent need for systematic safety evaluations.\u003c/p\u003e \u003cp\u003eCurrent safety assessments of MSC-EVs remain fragmented, predominantly focusing on acute toxicity endpoints or isolated immune parameters \u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Given the potential for organ accumulation and delayed immunogenicity, comprehensive characterization of cumulative biosafety risks following repeated MSC-EVs exposure is imperative.\u003c/p\u003e \u003cp\u003eTo address these gaps, we established a comprehensive safety assessment framework for MSC-EVs through systematic comparative analyses with polyethylene glycol (PEG)-liposomes, which served as a pharmacokinetic-matched controls. This framework enabled multidimensional evaluation of haemodynamics, biodistribution, and long-term safety profiles. We established a xeno-free production platform using human platelet lysate to generate clinical-grade hucMSC-EVs\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, eliminating animal-derived contaminants while maintaining EVs functionality. In vivo imaging system (IVIS) imaging revealed pharmacokinetic behavior: hucMSC-EVs exhibited hepatic and splenic tropism and 4-hour circulatory persistence. Our multilayered safety paradigm, including hematological analysis, cytokine storm risk assessment, multi-organ histopathology, and transcriptomics, showed that repeated administration of hucMSC-EVs induced significantly less inflammatory damage and immunogenicity compared to PEG liposomes. These findings validate the translational potential of hucMSC-EV as a low-risk therapeutic platform for regenerative medicine.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ehucMSC-EVs identification and adequate labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehucMSCs were isolated and expanded under xeno-free conditions using hPL as a serum substitute (Fig. S1A). Flow cytometry analysis confirmed the presence of mesenchymal stromal cell markers, including CD29, CD44, CD90, CD73, and CD105, while showing negative expression of CD45, CD11b, CD34, and HLA-DR in the isolated hucMSCs (Fig. S1B). Furthermore, trilineage differentiation assays validated their multipotency, as evidenced by osteogenic, adipogenic, and chondrogenic differentiation capabilities (Fig. S1C-E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehucMSC-EVs were isolated through sequential ultracentrifugation and size-exclusion chromatography (SEC),\u0026nbsp;and characterized in accordance with the International Society for Extracellular Vesicles (ISEV) 2023 guidelines\u003csup\u003e[19]\u003c/sup\u003e. Transmission electron microscopy (TEM) imaging revealed the characteristic cup-shaped morphology of hucMSC-EVs (Fig. 1A). Nanoparticle tracking analysis (NTA) \u0026nbsp;demonstrated a median diameter of 146.5\u0026nbsp;± 8.3 nm (Fig. 1B). Western blot analysis demonstrated expression of EV-associated markers (Alix, CD9, CD81, Tsg101), while excluding the endoplasmic reticulum (ER) marker Calnexin (Fig. 1C).\u0026nbsp;The results indicated that the positive rate of CD81 in hucMSC-derived EVs was 29.2%. In contrast, the positive rate of CD9 was 20.8% (Fig. 1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetics and biodistribution of hucMSC-EVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs natural nanocarriers of bioactive molecules, the circulatory persistence of hucMSC-EVs is critical for optimizing their therapeutic efficacy. To compare the\u0026nbsp;pharmacokinetic behavior\u0026nbsp;of DiD-labeled hucMSC-EVs with PEG-liposomes, we intravenously administered both formulations to 8-week-old C57BL/6 mice and serially monitored blood fluorescence intensity using the IVIS system‌‌.\u003c/p\u003e\n\u003cp\u003eFluorescence decay curves over time showed that hucMSC-EVs exhibited a blood half-life of approximately 3 h, whereas PEG-liposomes displayed prolonged circulation with a half-life of roughly 6 h. Both agents underwent rapid clearance, with almost complete elimination within 24 h (Fig. 2A). The delayed clearance of PEG-liposomes at 20 hours aligns with prior studies demonstrating that PEGylation enhances hydrophilicity and extends plasma residence time Notably, PEG-liposomes exhibited delayed clearance compared to hucMSC-EVs at 20 hours, consistent with previous reports that PEGylation improves hydrophilicity and prolongs in vivo circulation time \u003csup\u003e[20, 21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEx vivo biodistribution profiling demonstrated that hucMSC-EVs accumulated mainly in the liver, and PEG-liposomes mainly in the liver and spleen. (Fig. 2B, C). During the observation period, PEG-liposome accumulation was higher in the spleen than in hucMSC-EVs (Tab. S1, 2). Minimal signals were detected in the non-mononuclear phagocyte system (MPS) organs, including the brain and heart.\u003c/p\u003e\n\u003cp\u003eTo corroborate imaging data, tissue sections from the major accumulating organs were collected at 8 hours post-injection. These results are consistent with the overall organ imaging results showing that both PEG-liposomes and hucMSC-EVs were predominantly accumulated in the liver, with the spleen accumulating larger amounts of PEG-liposomes than hucMSC-EVs (Fig. 2D). Notably, fluorescence imaging of the intestine, a representative organ of the digestive tract, revealed negligible signal (Fig. S3A), indicating that EVs and liposomes injected intravenously do not significantly accumulate in the digestive tract. These findings underscore the liver as the primary site of hucMSC-EV accumulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultidimensional assessment of the safety of long-term injection of hucMSC-EVs from haematology, serum biochemistry, and cytokine/chemokine analyses.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the long-term biosafety of hucMSC-EVs, we established a repeated dosing regimen in BALB/c mice: low-dose (10⁹ particles/injection) and high-dose (10¹⁰ particles/injection) groups received intravenous administrations every 3 days for 30 days (10 total injections).\u0026nbsp;In vivo safety was assessed by haematology parameters, serum biochemistry, cytokine/chemokine analyses, histopathology, and RNA-seq after the mid-term (5th injection) and terminal (10th injection) injections (Fig. 3A).\u003c/p\u003e\n\u003cp\u003eBody weight trajectories remained stable across all groups, with variations \u0026lt;5g \u0026nbsp;from baseline (Fig. 3B). Final weight gain analysis revealed comparable growth patterns among cohorts, demonstrating no metabolic disruption induced by hucMSC-EVs (Fig. 3C). Notably, there were no mouse deaths in any of the groups during our observation period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnalysis of complete blood counts after the 5th injection showed that neither low nor high doses of hucMSC-EVs caused a significant elevation of inflammatory cells in the blood. In contrast, PEG-liposomes caused inflammation:\u0026nbsp;low-dose PEG-liposomes(L-Lipo) significantly elevated white blood cell counts (WBC)\u0026nbsp;compared to PBS control group (PBS: 4.93\u0026nbsp;±0.81\u0026nbsp;×10\u003csup\u003e9\u003c/sup\u003e/L, L-Lipo: 7.13\u0026nbsp;±\u0026nbsp;1.68\u0026nbsp;×10\u003csup\u003e9\u003c/sup\u003e/L, high-dose PEG-liposomes(H-Lipo): 5.47\u0026nbsp;±1.50\u0026nbsp;×10\u003csup\u003e9\u003c/sup\u003e/L,\u0026nbsp;\u0026nbsp;low-dose hucMSC-EVs(L-MSC EV): 3.74±1.47×10\u003csup\u003e9\u003c/sup\u003e/L, high-dose hucMSC-EVs(H-MSC EV): 3.15±0.81×10\u003csup\u003e9\u003c/sup\u003e/L).\u0026nbsp;The counts and proportions of intermediate(Mid) cell and granulocyte (GR) were also elevated in the PEG-liposomes group compared to the PBS control group\u0026nbsp;(Tab. 1). Similarly, after the 10th injection, neither group of hucMSC-EVs caused an elevation of inflammatory cells, whereas persistent WBC activation was observed in the H-Lipo groups(Tab. 2). Critical organ function biomarkers(hepatic enzymes: alanine aminotransferase (ALT) (Fig. 3D) and aspartate aminotransferase (AST) (Fig. 3E), renal parameters: serum creatinine (Scr) (Fig. 3F) and blood urea nitrogen (BUN) (Fig. 3G) remained unaltered across all groups. To investigate potential immune response, we quantified 16 cytokines/chemokines following long-term injections(Fig. 3H, I). CCL27(Fig. 3J), IL-10(Fig. 3K), and CCL22(Fig. 3L) were increased in hucMSC-EVs after five injections compared to the PBS group. Similar increases in both inflammatory factors were observed in the Liposomes group. In addition, CCL22, CCL12, CCL7, IL-6, and CCL11 showed an upward trend in the Liposomes group (all \u003cem\u003ep\u0026gt;\u003c/em\u003e0.05). After ten injections, no significant changes in inflammatory factors were observed in both groups. CXCL5 was up-regulated in the hucMSC-EVs group, and CCL27, IL-10, CCL1, IFN-g, IL-6, CXCL1, CCL17 in the Liposomes group (all \u003cem\u003ep\u0026gt;\u003c/em\u003e0.05). It is worth mentioning that we observed an upregulation of some inflammatory factors in the PBS group during long-term injections as well, which may be due to the endothelial damage of the veins caused by the repeated puncture of multiple injections, which activates platelets and immune cells to release inflammatory factors\u003csup\u003e[22, 23]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results suggest that long-term administration of hucMSC-EVs caused a lower risk of systemic inflammation compared to PEG-liposomes. Specifically, hucMSC-EV-treated cohorts exhibited stable white blood cell counts and minimal activation of inflammatory cytokine storms, underscoring their superior biocompatibility for chronic therapeutic applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology evaluation confirms favorable safety profile of hucMSC-EVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrgan sections collected after 10 intravenous injections were histologically assessed to evaluate potential tissue damage from long-term nanoparticle administration. Both hucMSC-EVs and PEG-liposomes exhibited no significant histopathological alterations in the brain, spleen, or heart, with all scores aligning with the PBS control group (score: 0) (Fig. 4A). This \u0026nbsp; absence of damage may be due to the minimal retention of PEG-liposomes and hucMSC-EVs in these organs, as shown by the organ imaging results mentioned earlier.\u003c/p\u003e\n\u003cp\u003eHepatocytes in PBS control group displayed characteristic radial plate arrangement around central veins, with intact sinusoidal structures.\u0026nbsp;hucMSC-EV-treated groups showed no significant hepatic damage, with sporadic inflammatory cell infiltration observed in a few mice, without dose-dependent progression. However, the H-Lipo group showed significant inflammatory infiltration compared to the PBS control group (\u003cem\u003ep\u0026lt;\u003c/em\u003e0.05). (Fig. 4A, B).\u003c/p\u003e\n\u003cp\u003eIn the pulmonary pathology, the PBS control group had intact tissue structure with normal alveolar morphology and no inflammatory reaction. The hucMSC-EVs group displayed \u0026nbsp;mild histopathological changes, (L-MSC EV group score:1.29±1.11, H-MSC-EV group score:0.86±1.07), as evidenced by alveolar wall thickening and inflammatory cell infiltration. In contrast, the L-Lipo group showed more severe damage, characterized by inflammatory cell infiltration, alveolar congestion, and alveolar wall thickening (score: 2.6±0.55) (Fig. 4A, C). Control glomeruli exhibited patent capillary loops without proteinaceous deposits. Following 10 injections, L-MSC EV and H-MSC EV groups demonstrated few scattered isolated foci (score: 0.43 ± 0.53), whereas H-Lipo induced significant inflammatory infiltrates (score: 1.2 ± 0.84) (Fig. 4A, D).\u003c/p\u003e\n\u003cp\u003eCollectively, continuous administration of hucMSC-EVs poses a lower risk of tissue and organ lesions compared to PEG-liposomes, affirming their enhanced organ safety profile for long-term therapeutic use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic profiling reveals organ-specific responses to hucMSC-EVs and PEG-Liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying tissue responses, transcriptome sequencing was performed on liver, lung, and kidney tissues from mice receiving 10 injections of hucMSC-EVs or PEG-liposomes. Principal component analysis (PCA) showed strong within-group clustering across all organs (Fig. S4A-C), confirming experimental reproducibility.\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis revealed 659 differentially expressed genes (DEGs) (327 upregulated, 332 downregulated) in hucMSC-EV-treated liver tissues, compared to 669 DEGs (492 upregulated, 177 downregulated) in PEG-liposome-treated counterparts. It is noteworthy that 18 inflammation-associated genes (major histocompatibility complex‌ class Ⅱ (H2-Aa), major histocompatibility complex, class II invariant chain (Cd74), myeloperoxidase (Mpo), chemokine (C-C motif) \u0026nbsp;ligand 5(Ccl5), serum amyloid A2(Saa2), etc.) were upregulated in PEG-liposome groups but remained unchanged or downregulated in hucMSC-EV groups (Fig. 5A). KEGG pathway analysis identified distinct enrichment patterns: both groups shared activation in pancreatic secretion, protein and fat digestion and absorption pathways, and MAPK signaling. However, PEG-liposomes uniquely activated immune and inflammatory pathways, including IL-17 signaling, antigen processing and presentation, and T-cell differentiation, whereas hucMSC-EVs modulated homeostatic pathways such as circadian rhythm, cardiac muscle contraction, and endocrine signaling pathways (e.g., cortisol and oxytocin) (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis revealed 659 differentially expressed genes (DEGs) (327 upregulated, 332 downregulated) in hucMSC-EV-treated liver tissues, compared to 669 DEGs (492 upregulated, 177 downregulated) in PEG-liposome-treated counterparts. It is noteworthy that 18 inflammation-associated genes (major histocompatibility complex‌ class Ⅱ (H2-Aa), major histocompatibility complex, class II invariant chain (Cd74), myeloperoxidase (Mpo), chemokine (C-C motif) \u0026nbsp;ligand 5(Ccl5), serum amyloid A2(Saa2), etc.) were upregulated in PEG-liposome groups but remained unchanged or downregulated in hucMSC-EV groups (Fig. 5A). KEGG pathway analysis identified distinct enrichment patterns: both groups shared activation in pancreatic secretion, protein and fat digestion and absorption pathways, and MAPK signaling. However, PEG-liposomes uniquely activated immune and inflammatory pathways, including IL-17 signaling, antigen processing and presentation, and T-cell differentiation, whereas hucMSC-EVs modulated homeostatic pathways such as circadian rhythm, cardiac muscle contraction, and endocrine signaling pathways (e.g., cortisol and oxytocin) (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eThe kidneys showed similar between-group differences to the lungs. There are 713 DEGs (308 upregulated, 405 downregulated) in hucMSC-EVs-treated kidneys, and 539 DEGs (247 upregulated, 292 downregulated) in PEG-liposomes-treated kidneys. Like the hepatic response pattern, hucMSC-EVs predominantly influenced steroidogenesis and lipid metabolism, while PEG-liposomes sustained the activation of immune pathways. In addition, hucMSC-EV-treated kidneys were consistently enriched in complement components, with lungs showing differential expression of 11 complement-related genes, such as complement component 6(C6), complement component 9(C9), alpha2-HS glycoprotein (Ahsg), and vitronectin (Vtn), in hucMSC-EV and PEG liposomes. hucMSC-EVs consistently upregulated complement and coagulation cascades in lung and kidney tissues (Fig. 5E, F). This coordinated regulation of interconnected homeostatic systems suggests a potential immunomodulatory mechanism for hucMSC-EVs biocompatibility.\u003c/p\u003e\n\u003cp\u003eConsistent with hematological and histopathological findings, transcriptomic signatures indicate that long-term hucMSC-EVs injections elicit significantly lower inflammatory pathway activation than PEG-liposomes across multiple organ systems.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe core bottlenecks in the current clinical application of MSC-EV are fluctuations in therapeutic efficacy due to heterogeneous sources, insufficient target delivery efficiency, and, more importantly, the lack of comprehensive safety assessment and quality control standards to ensure therapeutic efficacy and avoidance of side effects and to promote the development of clinical applications. Our study evaluated the safety of hucMSC-EVs in terms of biodistribution, blood cytology assay, serum biochemistry assay, serum inflammatory factors and chemokines assay, pathological damage score, and RNA-seq.\u0026nbsp;It demonstrated that it does not cause significant inflammatory response and tissue damage and has a good safety profile for long-term administration.\u003c/p\u003e \u003cp\u003eWe injected mice with DiD-labelled hucMSC-EVs and PEG-liposomes, using serum and organ imaging acquired at different time points after injection. We found that hucMSC-EVs were metabolised slightly faster than PEG-liposomes, but the metabolic trends of both were highly similar. This parallelism suggests that despite the diverse biological origins and molecular compositions of nanoscale carriers, whether endogenous EVs or synthetic liposomes, their systemic fates may be governed by common biophysical principles. Whereas, existing studies have clarified that the blood residence time and organ targeting efficiency of nanoparticles are mainly regulated by the following three physical factors, including EV size, surface charge, and membrane fluidity\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Among these, nanoparticle size is the major influencing factor, affecting their uptake, biodistribution, and circulating half-life. The bEVs (\u0026gt;\u0026thinsp;200 nm) and sEVs (\u0026lt;\u0026thinsp;200 nm) of invasive triple-negative breast cancers predominantly target the lungs, liver, and spleen (sEV only) at low doses, and sEVs are more readily transported from other major organs to the liver over time\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Smaller-sized EVs can more easily penetrate the vascular endothelial barrier and have unique biodistribution properties \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In addition, during culture and after entering the somatic circulation, EVs are exposed to various proteins that bind to the EVs to form protein crowns, which in turn affect the behaviour of the EVs\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. It was found that pre-coating EVs with albumin to create albumin-rich protein corona-EV complexes enhanced non-phagocytic uptake in the liver\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, a phenomenon that could be used as a camouflage strategy to enhance EV targeting.\u003c/p\u003e \u003cp\u003eBeyond these inherent properties, emerging engineering strategies can further reprogram EV biodistribution. Targeted delivery of EVs can also be facilitated by altering the in vivo distribution of EVs by adding homing peptides to the EV surface. For example, peptides such as RGD and its cyclic variant c(RGDyK) bind target cells\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, while cardiomyocyte-specific peptides (CMP) guide EVs to heart tissue\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Other reported sequences include ASSLNIA and CSTSMLKAC (targeting muscle and ischemic myocardium), a series of T140 variants (T140, Scr‑T140, T140‑KLA, B‑TL5) directed at CXCR4‐expressing cells\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, PTHTRWA for lung cancer targeting, an EGFR‑targeting peptide\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, CKBP for aneurysmal smooth muscle cells, and a Lys‑Leu‑Ala peptide used with low‑density lipoprotein mimetics for glioma\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In addition, modification of extracellular vesicle glycosylation can alter their in vivo distribution. The investigators treated EVs from mouse liver proliferating cells with neuraminidase to digest the glycoprotein-terminal sialic acid residues on the surface of the EVs. They found that the modified EVs induced accumulation in the lungs when injected intravenously, and additionally, when injected through the tarsal joints, the neuraminidase-treated vesicles were better distributed in the axillary lymph nodes than the untreated EVs\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results of our multidimensional safety assessment based on MSC-EVs showed a clear safety profile of MSC-EVs.The superior safety profile of MSC-EVs compared to MSCs is since they do not express major histocompatibility complex (MHC) class I/II antigens on their surfaces, which considerably reduces the risk of immune rejection and tumour formation\u0026zwnj;\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In vitro toxicological studies have demonstrated that MSC-EVs are safe, with no detectable genotoxic effects, haemolysis, or significant effects on platelet aggregation\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Available clinical trials have shown that MSC-EVs are safe for various diseases with few adverse effects\u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. However, existing safety evaluations remain fragmented, often neglecting dose-dependent responses, immunogenicity, and mechanistic insights.\u003c/p\u003e \u003cp\u003eThe basic factors affecting nanoliposome safety include composition, size, surface charge, stability, release of dopant drug, and penetration into tissues \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. The advantage of cationic surface liposomes is that they can bind negatively charged cargoes through electrostatic interactions. Although unmodified cationic liposomes have no or minimal toxicity at low doses, some cytotoxic effects have been reported. For example, cationic liposomes are toxic when administered in multiple doses, whereas neutral liposomes are relatively safe. Studies have shown no change in mortality in mice in the long-term treatment group with neutral liposomes. In contrast, the mortality rate of mice in the long-term treatment group with positively charged liposomes was 45 percent\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Furthermore, positively charged liposomes lead to more severe changes in blood levels and inflammatory components than neutral liposomes. This may be due to surface positively charged groups inducing ROS production in cells \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and cationic liposomes interacting with the plasma membrane of neutrophils and triggering neutrophil stimulation\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Polyethylene glycolisation effectively prolongs the systemic circulation time of LNPs, thereby greatly improving their pharmacokinetics and efficiency\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. However, the novel PEG liposomes are not completely non-toxic and may present a risk of cytotoxic tissue oxidative damage\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Repeated injections of PEG liposomes may cause an unexpected immunogenic response, producing antibodies to PEG and leading to accelerated blood clearance (ABC) \u003csup\u003e[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. In addition, side effects such as acute infusion reactions and hand-foot syndrome (HFS) induced by liposomal drugs cannot be ignored, as documented by clinical studies \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Our finding revealed that long-term injection of PEG liposomes did not cause death in mice, but an increase in blood leukocyte counts was observed in the L-Lipo group and the H-Lipo group after 5 sustained injections, and in the H-Lipo group after 10 injections, which is consistent with other literature reporting that liposomes may cause blood inflammation. Pathological damage in long-term injections of PEG-Liposomes was mainly concentrated in the liver and lungs. Still, the pathological damage scores were less than or equal to 3 for all organs (the most severe damage was 4). In addition, long-term injection of PEG-Liposomes caused activation of hepatic, pulmonary, and renal IL-17 signalling, T-cell differentiation, TLR pathway, and other immune and inflammation-related pathways in mice.\u003c/p\u003e \u003cp\u003eOur comprehensive safety evaluation revealed that hucMSC-EVs have a good safety profile even after long-term injections, with three significant findings: 1. hucMSC-EVs have a faster blood clearance rate than PEG-liposomes, and their main organ of distribution in the body is the liver; 2. hucMSC-EVs are highly biocompatible, and long-term injections cause less immune activation; 3. hucMSC-EVs transcriptome features show an enrichment of metabolic homeostasis rather than inflammatory responses, in addition to the unique complement-coagulation cascade regulation of hucMSC-EVs. hucMSC-EVs have better biocompatibility and less immune activation caused by long-term injection; 3. hucMSC-EVs transcriptome features show enrichment in metabolic homeostasis rather than inflammatory response, and in addition, the unique complement-coagulation cascade regulation of hucMSC-EVs may be responsible for the immune tolerance mediated by hucMSC-EVs. Comprehensive safety assessment of MSC-EVs is not only about individual therapeutic risk control, but also a core aspect of translation from basic research to clinical application. In conclusion, our study demonstrated the safety of long-term administration of hucMSC-EVs, and laid a sure foundation for the future development of MSC-EVs as drug delivery vehicles and clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82230022, 82030024, 81720108007), the National Key Research and Development Program of China (2022YFC2502500), the Joint Innovation Laboratory for the Industrialization of Extracellular Vesicle-Based Drug Delivery Technology Established by Shenzhen Kexing Pharmaceutical Co., Ltd. and Southeast University (8524006050) to B.-C.L. This research was supported by additional grants from National Natural Science Foundation of China (82200772), the Natural Science Foundation of Jiangsu Province grant (BK20220828), the Fundamental Research Funds for the Central Universities (2242023R40030), and the Start-up Research Fund of Southeast University (RF1028623235) to T.-T.T. It is also supported by Shenzhen Major Science and Technology Project (KJZD20230923115201003) and Beijing Echo Biotech Co., Ltd.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ehucMSC Extraction\u003c/strong\u003e\u003cstrong\u003eand culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;umbilical cord donors provided their informed consent. All procedures were approved by the Ethical Committee for Clinical Research of Zhongda Hospital, Affiliated to Southeast University (Approval No. 2023ZDSYLL118-P01). After rinsing the umbilical cord twice with pre-cooled sterile PBS, the amniotic membrane, two arteries, and one vein were removed with forceps, and the remaining umbilical cord tissue was cut into pieces of 1-3 mm\u003csup\u003e3\u003c/sup\u003e with a piece of Warton's glue. The pieces of umbilical cord tissue were transferred into a 50 mL centrifuge tube, 20 mL of PBS was added, and centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was discarded, and the pre-configured complete medium(base medium(MSC NutriStem® XF Medium, 05-200-1A, Sartorius)containing 5% hPL (PLTGOLD100R, Sartorius) )for hucMSCs was added, and the medium containing the pieces of umbilical cord tissue was transferred into a T75cm\u003csup\u003e2\u003c/sup\u003e cell culture flask. Cultivate the cells at 37℃\u0026nbsp;in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, and change the medium half every three days, and cells can be seen crawling out around the tissue mass in about one to two weeks. When the cell fusion around the tissue block reaches 80%-85%, the cells can be passed on. After the cells were washed with sterile PBS once, add 3ml TrypleLE Express (12604039, Gibico), digest the cells for about 2 min, add an equal amount of fresh medium to neutralize, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh complete medium and mix gently by blowing, and then transfer to new culture flasks for passaging culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehucMSC-EVs Isolation, Purification, and Storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen the hucMSCs grew to 75%-80% fusion, discard the old medium and wash the cells twice with PBS; add the basal medium DMEM/F12(Gibico, 11320033) and incubate the cells in a 37°C, 5% CO2 incubator for 48 h. Collect the supernatant into a centrifuge tube and centrifuge the cells at 4 ℃ for 20 min at 2,000 g. Transfer the supernatant into another centrifuge tube and centrifuge the supernatant at 4 ℃ and 13,500 g for 30 min. The supernatant was transferred to another centrifuge tube and centrifuged at 4 ℃ and 13500 g for 30 min; the supernatant was taken into an ultracentrifuge tube and ultracentrifuged at 4 ℃ and 100000 g for 2 h. The precipitates were resuspended by adding an appropriate amount of sterile PBS filtered through a 0.22 μm filter to obtain the preliminary extracted hucMSC-EVs. The size exclusion chromatography (SEC) Exo exclusion column(Echo9101A-S10, Echo Biotech) was equilibrated to room temperature, 20 mL of PBS was used to rinse the column, 1 mL of preliminary extracted hucMSC-EVs was added, and when all of the sample was in the separation column, PBS was added for elution, 500 ul for 1 fraction, and the EV-enriched fractions 3-7 were collected after purification.\u0026nbsp;hucMSC-EVs were freshly extracted and stored at −80 °C for no more than one month before usage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehucMSC-EVs Detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurface marker profiling was performed through immunoblotting analysis. Briefly, exosome-specific proteins (Alix [ab117600, Abcam], CD9 [ab236630, Abcam], CD81 [ab97559, Abcam], Tsg101 [ab125011, Abcam]) and the endoplasmic reticulum marker Calnexin (ab22595, Abcam) were probed using Western blot. For nanoparticle characterization, EV samples were subjected to ExoView™\u0026nbsp;platform (NanoView Biosciences, Boston, MA) analysis following the manufacturer's protocol. Prior to immunofluorescence staining, EV concentrations quantified via nanoparticle tracking analysis (NTA) were adjusted to 1–3 × 10⁸ particles/mL by 1:1 dilution with incubation buffer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNano flow cytometry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLoad 30ul hucMSC(10\u003csup\u003e10\u003c/sup\u003e) into EP tube for each component, add 1 test amount of antibody(CD9,BD Pharminge,555371) (CD81,BD Pharminge,551108) according to the antibody instruction, mix thoroughly, and incubate for 1 hour at 37℃, protected from light. After incubation, add 1ml of 0.22 um filtered PBS to transfer the EV to the ultra-isolated tube, and then ultracentrifuge at 4℃, 100000g for 75min after leveling. Avoid light as much as possible and resuspend the EV with 30-50 ul PBS in a 600 ul EP tube. Power on the nanofluidizer and start the system, after completing the routine maintenance and quality control, put the stained samples on the sample stage, turn on the switch of the fluorescence channel that the sample is to be detected, and then the software will automatically set the threshold value, collect and save the sample data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescent labeling and free dye purification of hucMSC-EVs and Liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe labeled hucMSC-derived extracellular vesicles (EVs) and PEG-liposomes with 1 mM of DiD dye (C1039, Beyotime) and incubated them at room temperature in the dark with a shaking speed of 40 rpm for 2 hours. Subsequently, unbound free dye was removed using size-exclusion chromatography (SEC) as previously described. Preliminary experiments involved collecting fractions 1-15, where we assessed the fluorescence intensity of different fractions using a multifunctional microplate reader (Fig. S2A). Additionally, electron microscopy analysis (Fig. S2D) and Western blot (WB) detection of the EV markers were performed to confirm that EVs were predominantly enriched in fractions 4-7. Consequently, we pooled fractions 4-7 for further analysis. Finally, we conducted a preliminary evaluation of the fluorescence positivity rate of the labeled liposomes and hucMSC-EVs using nanoparticle tracking analysis to assess the impact of DiD labeling (Fig. S2E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood metabolism and distribution imaging after MSC-EVs or PEG-liposomes injection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old C57BL/6J mice\u0026nbsp;(purchased for the experiments from: Beijing Vital River Laboratory Animal Technology Co., Ltd., China) weighing 20-23 g were selected and fasted for 12 h. Mice were injected into the tail vein with 200 uL of 1×1010 particles DiD-labeled MSC-EVs, PEG-liposomes, or PBS. Mice were anaesthetised by intraperitoneal injection of tribromoethanol at a concentration of 1.25% at a dose of 200 ul per 10 g of mouse body weight, and the blood was collected from the heart into a 1.5 mL EP tube and left to stand at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min, After injection, mice were executed at different time points: 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, and 72h, and blood was collected from the heart into 1.5mL EP tubes, which were left at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min, and the upper layer of the serum was removed, and the brain, heart, liver, lungs, spleen, kidneys, and gastrointestinal tract were retained, and the serum and the organisms were subjected to optical imaging, respectively. Fluorescence values were normalized to the 0.5-hour baseline measurement to calculate relative blood retention percentages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoutine blood tests and biochemistry testing of blood samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples for routine blood tests were added to 1.5 mL EP tubes pre-filled with anticoagulant(YA1461, Solarbio). The automatic blood cell analyzer( DF62 lab, DYMIND) starts automatic measurement after setting the species.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of serum cytokines by liquid-phase suspension microarray technology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobeads were shaken with a shaker, at 1,400 rpm, for 30 seconds and then 50 ul per well was added to the 96-well plate. After 4-fold dilution of each sample, take 50ul of the sample, the standard and the blank control were prepared according to the instructions, also take 50ul of the sample, affixed with a sealing film, and placed on a plate shaker at 850 rpm, protected from light, at room temperature, and incubated for 30 min. Discard the sample, use a plate washer to wash it 3 times, and then dilute it using the Antibody Diluent according to the instructions. DetectionAntibody, 25 μL of diluted DetectionAntibody was added to each well, sealed with a sealing film, and incubated on a plate shaker with shaking at 850 rpm, protected from light, and at room temperature for 30 min. Samples were discarded, washed 3 times with a plate washer, and diluted with Assay Buffer as described in the instructions. Streptavidin-PE, add 50 μL of diluted Streptavidin-PE to each well, affix a sealing film, and incubate for 10 min at 850 rpm on a plate shaker with shaking, protected from light, and at room temperature. Wash three times with a plate washer, and then resuspend with 125 μL of Assay Buffer to each well, affix a sealing film, and incubate for 30 min at 850 rpm, room temperature, and protected from light. 850 rpm, room temperature, protected from light, shaken for 30 s and placed in a calibrated Bio Plex 200 machine to read the values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin-Eosin staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHE staining was performed using 3μm thick paraffin-embedded tissue sections. Tissue sections were placed in a thermostat box and baked at 60°C for 1 h. Sections were deparaffinized twice in a xylene vat for 15 min each time, then sequentially placed in a gradient ethanol vat for hydration, and the sections were rinsed in ddH2O for 5 min. Sections were placed in Hematoxylin Staining Solution for 10 min, slowly rinsed in ddH\u003csub\u003e2\u003c/sub\u003eO for 10 min, decolorized in hydrochloric acid-alcohol for 20 s, and rinsed in ddH\u003csub\u003e2\u003c/sub\u003eO for 10 min, and returned to blue in ammonia water for 3 min. After returning to blue for 3 min, ddH\u003csub\u003e2\u003c/sub\u003eO was rinsed for 10 min, dehydrated in 70% and 90% alcohol for 10 min each, staining with eosin staining solution for 2-3 min, dehydration in gradient ethanol, followed by xylene transparency for 5 min, air drying, and sealing with drops of neutral gum and coverslips.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological damage score\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystematic histomorphological evaluation was performed on paraffin-embedded sections of six major organs following 5 and 10 times injections. Tissue injury was quantified using a 5-tier histopathological scoring system (0: intact microstructure;\u0026nbsp;1 : slight damage (occasional cellular degeneration or inflammation); 2 : mild damage (localized cellular degeneration or mild inflammation); 3 : moderate damage (marked cellular degeneration or moderate inflammatory cell infiltration) 4: severe necrosis/architectural disruption (extensive cellular necrosis or severe disruption of tissue structure)).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted with TRIzol, and then the purity and concentration of RNA were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Transcriptome libraries were constructed using the VAHTS Universal V5 RNA-seq Library Prep Kit and sequenced using the llumina Novaseq 6000 sequencing platform. Reference genome comparisons were performed using HISAT2 software, gene expression (FPKM) calculations were performed, and read counts (counts) for each gene were obtained by HTSeq-count. PCA analysis of genes (counts) as well as mapping was performed using R (v 3.2.0) to assess sample biological replicates. Differentially expressed genes were analyzed using DESeq2 software, where genes that met the thresholds of q-value \u0026lt; 0.05 and foldchange \u0026gt; 2 or foldchange \u0026lt; 0.5 were defined as differentially expressed genes (DEGs). Hierarchical clustering analysis of DEGs was performed using R (v 3.2.0) to demonstrate the expression patterns of genes across groups and samples. Radar plots were created for the top 30 genes using the R package ggradar to demonstrate changes in expression of up- or down-regulated genes. Subsequently, GO enrichment analysis of differentially expressed genes based on the hypergeometric distribution algorithm was used to screen for significantly enriched functional entries. R (v 3.2.0) was used to plot bar charts, chord charts or enrichment analysis circle plots for significantly enriched functional entries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were expressed as mean\u0026nbsp;±SD. Statistical analysis was performed using a twotailed Student’s t test or one-way analysis of variance (ANOVA), with P \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003econsidered significant.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJEPPESEN D K, FENIX A M, FRANKLIN JL et al. Reassessment Exosome Composition [J] Cell, 2019, 177(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRANI S, RYAN A E, GRIFFIN M D, et al. 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Membrane Protein Modification Modulates Big and Small Extracellular Vesicle Biodistribution and Tumorigenic Potential in Breast Cancers In Vivo [J]. Adv Mater. 2023;35(13):e2208966.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eABRA R M, HUNT CA. Liposome disposition in vivo. III. Dose and vesicle-size effects [J]. Biochim Biophys Acta. 1981;666(3):493\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSOO C Y, SONG Y, ZHENG Y, et al. Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells [J]. Immunology. 2012;136(2):192\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXIAO Q, ZOULIKHA M. The effects of protein corona on in vivo fate of nanocarriers [J]. Adv Drug Deliv Rev. 2022;186:114356.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHEIDARZADEH M, ZAREBKOHAN A, RAHBARGHAZI R, et al. Protein corona and exosomes: new challenges and prospects [J]. 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Pharmaceuticals (Basel), 2022, 15(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eABU LILA A S, KIWADA H, ISHIDA T. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage [J]. J Control Release. 2013;172(1):38\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eISHIDA T, ATOBE K, WANG X, et al. Accelerated blood clearance of PEGylated liposomes upon repeated injections: effect of doxorubicin-encapsulation and high-dose first injection [J]. J Control Release. 2006;115(3):251\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eISHIDA T, KASHIMA S, KIWADA H. The contribution of phagocytic activity of liver macrophages to the accelerated blood clearance (ABC) phenomenon of PEGylated liposomes in rats [J]. J Control Release. 2008;126(2):162\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePETERSEN G H, ALZGHARI S K, CHEE W, et al. 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J Control Release. 2016;232:255\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"extracellular vesicles, MSC, PEG-Liposome, safety, long-term injection","lastPublishedDoi":"10.21203/rs.3.rs-6931168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6931168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMesenchymal stem cell-derived extracellular vesicles (MSC-EVs) hold significant therapeutic promise in regenerative medicine; however, their long-term safety profile remains inadequately characterized for clinical translation. In this study, human umbilical cord MSC-derived EVs (hucMSC-EVs) were produced using a heterologous component-free platform by substituting foetal bovine serum (FBS) with human platelet lysate (hPL). A comprehensive safety assessment was conducted by benchmarking hucMSC-EVs against polyethylene glycol (PEG)-liposomes. Pharmacokinetic analysis via cell membrane red fluorescent probe (DID)-labelling revealed rapid clearance of hucMSC-EVs, with a blood half-life of approximately 3 hours and predominant hepatic accumulation. Multi-dose evaluations integrating hematological profiling, cytokines/chemokines quantification, and histopathological scoring demonstrated superior biocompatibility of hucMSC-EVs over PEG-liposomes. Key findings include: 1. hucMSC-EVs did not induce activation of blood inflammatory cells; 2. hucMSC-EVs caused a significant increase in CCL12 only, and the rest of the multiple inflammatory factors/chemokines remained stable; 3. histopathological changes associated with hucMSC-EVs were mainly localized to liver and lungs, yet markedly less severe than PEG-liposome-induced lesions; 4. Transcriptome sequencing further demonstrated that hucMSC-EVs regulated metabolism and complement-coagulation cascade while avoiding activating pro-inflammatory pathways. In contrast, PEG-liposomes up-regulated inflammation-associated pathways such as IL-17 signalling, the MAPK pathway, the TLR pathway, and the T-cell differentiation pathway. 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