Exosome-Biomimetic Nanoprobes Achieve Ultrasensitive Detection and Real-Time Monitoring of Liver Metastasis

Exosome-Biomimetic Nanoprobes Achieve Ultrasensitive Detection and Real-Time Monitoring of Liver Metastasis | 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 Exosome-Biomimetic Nanoprobes Achieve Ultrasensitive Detection and Real-Time Monitoring of Liver Metastasis Zuoyu Xu, Hongyan Zou, Lixin Cheng, Xiaoqiu Dong, Daoshuang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7556946/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2026 Read the published version in Cancer Nanotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Background ​​: Liver metastasis (LM) remains a leading cause of cancer mortality, with conventional imaging modalities suffering from physiological interference and insufficient sensitivity. Fluorine-19 (¹⁹F) MRI offers a promising solution due to its negligible endogenous background, yet existing targeted probes face limitations in tumor heterogeneity and receptor variability. ​​ Methods ​​: We developed exosome-biomimetic nanoparticles (EP-NPs) by coating perfluorocarbon nanoparticles (PFCE NPs) with exosomal membranes derived from HEK-293T cells. The hybrid platform combined the targeting capability of exosomes with the high sensitivity of ¹⁹F MRI. Characterization included dynamic light scattering, transmission electron microscopy, and ¹⁹F NMR. In vitro cellular uptake was evaluated in BT-549 and NCI-H446 cells using confocal microscopy. In vivo targeting efficiency and biodistribution were assessed in a BT-549 liver metastasis mouse model via ¹⁹F/¹H MRI. Toxicity was tested via MTT assays, serum biochemistry, and histopathology. ​​ Results ​​: EP-NPs exhibited a hydrodynamic diameter of 111.6 ± 8.2 nm, negative zeta potential (-20.50 mV), and stable ¹⁹F signal. Confocal imaging confirmed enhanced cellular uptake compared to non-targeted PFCE NPs. In vivo , EP-NPs enabled precisely and early detection of LM with sustained ¹⁹F signal. Biodistribution revealed accumulation in the liver and spleen, and toxicity assessments demonstrated no significant hepatorenal impairment or histological damage. ​​ Conclusion ​​: EP-NPs integrate exosomal targeting and ¹⁹F MRI to achieve ultrasensitive LM detection with high biocompatibility and prolonged circulation. This platform holds potential for real-time monitoring of metastatic progression and clinical translation, overcoming limitations of conventional imaging agents. Liver metastasis ¹⁹F MRI Exosome Perfluorocarbon nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Hepatic metastasis persists as the principal contributor to cancer-related mortality worldwide (Wan, Pantel, and Kang 2013 ). The unique hepatic microenvironment, characterized by extensive vascularization and sinusoidal tissue architecture, provides an optimal niche for circulating tumor cell colonization, resulting in frequent liver metastasis (LM) across multiple malignancies (Milette et al. 2017 ). Detection of LMs constitutes a critical determinant for therapeutic outcomes. In clinical practice for detecting hepatic metastases, current imaging modalities primarily include computed tomography (CT), ultrasonography, and magnetic resonance imaging (MRI). Notably, gadolinium-based MRI demonstrates superior detection sensitivity among these techniques (Renzulli et al. 2020 ). Nevertheless, conventional proton-weighted MRI encounters inherent limitations including substantial physiological interference from endogenous water protons and carbohydrate derivatives (Berkova et al. 2022 ; Patil et al. 2011 ). To circumvent these constraints, fluorine-19 ( 19 F) MRI has emerged as an innovative molecular imaging strategy (Zhang et al. 2022 ; Havlicek et al. 2024 ). This modality capitalizes on the negligible endogenous fluorine background in biological systems, enabling quantitative hotspot imaging of exogenously administered fluorinated probes with enhanced signal specificity (Ruiz-Cabello et al. 2011 ). Perfluorocarbon nanoparticles (PFC NPs) functionalized with targeting ligands have shown promise in tumor-specific 19 F MRI applications (Xu et al. 2018 ; Sun et al. 2022 ). However, tumor heterogeneity and receptor expression variability across cancer subtypes constrained the clinical translation of these imaging agents that targeting only a single site (Labrijn et al. 2019 ; Lim et al. 2021). Recent advances in extracellular vesicle engineering have highlighted exosomes as promising theranostic vectors (Zeng et al. 2023 ; Lucchetti et al. 2023 ). Exosomes refer to a group of extracellular vesicles naturally released from mammalian cells with a diameter of 30–150 nm (Shao et al. 2025 ). Exosomes, functioning as nanoscale signaling vesicles, carry diverse nucleic acids and proteins, facilitating intercellular communication. Their membranes are embedded with various functional proteins that perform specific roles. What is more important, exosomes possess inherent cell-targeting capabilities due to the presence of specific membrane proteins, including tetraspanins and integrins (Yang et al. 2018 ; Ma et al. 2020 ; Zhang et al. 2020 ). Additionally, the ability of exosomes to shuttle molecules has facilitated their use as alternative vehicles for delivering molecular imaging agents within their lumen, transferring them to cells at distant sites in the body (Srivastava et al. 2022 ). All these distinctive characteristics position exosomes as exceptional nanoplatforms for imaging applications. In this investigation, we engineered a novel exosome-biomimetic nanoprobe named EP-NPs, using an exosomal membrane coated PFC NPs to achieve efficient and precise imaging of LMs. Systematic evaluation of tumor-targeting efficiency, biosafety profiles, and imaging performance revealed that the exosome-coated nanoplatform possess excellent tumor accumulation efficacy, high biocompatibility and sustained 19 F signal persistence.This nanoplatform generates a strategy for real-time monitoring and early detection of LMs. 2. Materials and Methods 2.1. Materials Cell culture media (RPMI-1640, Leibovitz’s L-15, DMEM), fetal bovine serum (FBS), and Trypsin-EDTA (0.25%) were supplied by Thermo Fisher Scientific Inc. (SHH, CHN). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent and dimethyl sulfoxide (DMSO) were procured from Sigma-Aldrich (SHH, CHN) and Tansoolereagent (SHH, CHN), respectively. Human cell lines, BT-549 (breast carcinoma), NCI-H446 (lung carcinoma), and HEK-293T (embryonic kidney)were acquired from Procell Life Science & Technology Co., Ltd. (HB, CHN). Perfluoro-15-crown-5-ether (PFCE, C 10 F 20 O 5 ) was sourced from Exfluor Research Corporation (TX, USA), while lecithin and cholesterol (Avanti Polar Lipids, AL, USA) and glycerin (Aldrich, WI, USA) constituted the lipid components. Ultra-high-purity argon (99.99%) was utilized for material storage. Dialysis membranes (20 kDa MWCO, Spectrum Medical Industries, CA, USA) and analytical-grade chemicals were employed without further purification. 2.2. Cell culture BT-549 and NCI-H446 cells were cultured in RPMI-1640 (supplemented with 10% FBS), HEK-293T was cultured in DMEM (supplemented with 10% FBS), all cells were cultured in a humidified atmosphere with 5% CO 2 at 37℃ and passed around 80% confluence. 2.3. Preparation of PFC NPs PFC NPs were prepared as per prior protocols (Wu et al. 2018 ). The emulsion comprised 20% (v/v) PFCE, 2% (w/v) surfactant blend (85 mol% lecithin, 2.9 mol% phosphatidylglycerol, 12 mol% cholesterol), 2.5% (w/v) glycerin, and water. Lipid films were formed by dissolving components in methanol-chloroform, evaporating solvents via rotary evaporation (45°C), and vacuum drying (24 h). Hydration with purified water, followed by vortexing (2 min) and probe sonication (2 min), yielded nanoparticle suspensions. Final emulsions were dialyzed (20 kDa MWCO cellulosic membrane), argon-purged, and stored at 4°C. 2.4. Preparation of exosome membrane coated PFC NPs Exosomes were harvested by collecting the supernatant from HEK-293T cell cultures grown in T225 flasks at 80% confluency, followed by isolation using differential centrifugation as described in previous studies (Zhan et al. 2020 ; Tian et al. 2014 ). All procedures were performed at 4°C. Initially, the exosome-containing supernatant was centrifuged at 3000g for 30 min to eliminate dead cells and debris. Subsequently, the supernatant underwent centrifugation at 10,000g for 45 min to remove larger vesicles. The clarified supernatant was then filtered through a 0.22 µm membrane (Millipore, USA). Exosomes were pelleted via ultracentrifugation at 100,000g for 90 min, resuspended in PBS, sonicated, and passed through a 200 nm microporous membrane to achieve a homogeneous suspension. The contents of exosomes were removed by hypotonic treatment and ultracentrifugation (Cheng et al. 2018 ). For exosome membrane-coated perfluorocarbon nanoparticles, empty exosome vesicles were combined with PFCE NPs and extruded 10 times through a 100 nm polycarbonate membrane using an Avanti mini extruder, yielding exosome-coated nanoparticles (EP-NPs). 2.5. Characterization of nanoparticles The hydrodynamic diameter,and zeta potential (ζ) of EP-NPs were analyzed using a Malvern Nano ZS Zetasizer (Malvern Instruments Ltd, UK) via dynamic light scattering (DLS). All measurements were repeated three times for consistency.The morphology of nanoparticles was observed with a Talos F200s transmission electron microscope (FEI, ThermoFisher, Waltham, MA) operating at 200 kV. Elemental mapping images were also obtained by using transmission electron microscopy (TEM). 19 F NMR characterization was performed using a 9.4T Bruker Avance III 400 MHz spectrometer. Spectra were acquired using a “single-pulse” sequence (Bruker“zg”sequence), with a D 2 O-filled capillary tube for field locking. Measurements were taken at -75.4 ppm, with a 50 ppm sweep width, 8 scans, and a 25-second interscan delay. Data processing was conducted by using Mestrenova 10.0.2. 2.6. Phantom experiments EP-NPs were dispersed in 1.7% agarose at PFCE concentrations of 4.8, 7.2, 9.6, 14.4, and 19.2 mmol/L and loaded into tubes. 19 F MR imaging was performed using a 9.4T small animal scanner (BioSpec 94/20 USR, Bruker, Germany) with a multi-slice multi-echo (MSME) sequence, featuring 24 echo times (TE) from 8 to 192 msec at 8-msec intervals. Imaging parameters included: repetition time (TR), 3000 msec; matrix size, 64×64; field of view (FOV), 38.4×38.4 mm; and slice thickness, 1 mm. 2.7. Cellular imaging experiment of EP-NPs BT-549 and NCI-H446 cells were plated in confocal microscopy dishes at a density of 1×10 5 cells per dish. Following a 24-hour incubation, the cells were treated with either EP-NPs (40 µL/mL culture media) or non-targeted PFCE NPs (40 µL/mL culture media). The PFCE nanoparticles in EP-NPs and non-targeted PFCE NPs were labeled with PKH67 and Rhodamine, respectively. After a 4-hour incubation at 37°C, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Subsequent to three PBS washes, the nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI). The dishes were then washed three more times, and cellular uptake was observed by confocal laser scanning microscopy (Nikon, Japan). 2.8. Cytotoxicity assay of EP-NPs The cytotoxicity of EP-NPs was assessed using the MTT assay. BT-549 and NCI-H446 cells were seeded in 96-well plates at 1×10 4 cells/well and incubated for 24 hours. Cells were then treated with varying concentrations of EP-NPs (PFCE concentrations were 4.8, 7.2, 9.6, 14.4, and 19.2 mmol/L) in a final volume of 200 µL/well and incubated overnight at 37°C. Following this, 20 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 4 hours. The medium was then replaced with 150 µL of DMSO, and absorbance at 490 nm was measured using a plate reader to determine cell viability. 2.9. Animal models Female BALB/c nude mice, aged 6–8 weeks, were sourced from Vital River Laboratory Animal Technology Co., Ltd. (BJ, CHN) and housed in specific pathogen-free conditions with unrestricted access to food and water. For the liver metastasis model, BT-549 cells were cultured and suspended in sterile PBS. Mice were anesthetized with isoflurane and positioned supine. A small left flank incision was made to expose the spleen using a retractor. A suspension of 1×10 6 BT-549 cells in 50 µL PBS was injected into the spleen parenchyma. The spleen was then returned to the abdominal cavity, and the incision was stitched up with a 5 − 0 absorbable suture 2.10. In vivo liver metastasis targeting and biodistribution studies. At the predetermined time points, mice were anesthetized with isoflurane and imaged using a 9.4T MR scanner (BioSpec 94/20 USR, Bruker) with a 1 H/ 19 F dual-tune volume coil. For in vivo tumor targeting study, 1 H (T2-weighted) and 19 F images were obtained from mice in the EP-NPs group (100 µL/mouse) and the non-targeted PFCE NPs group (100 µL/mouse) before and after intravenous injection. 1 H imaging utilized a rapid acquisition with relaxation enhancement (RARE) sequence with the following scan parameters: TR/TE = 3000/40 msec, NA = 2, RARE factor = 10, matrix = 256×256, FOV = 38.4×38.4 mm², and slice thickness = 1.0 mm. The 19 F RARE sequence, aligned with the proton images, was acquired with TR/TE = 2000/100 msec, NA = 128, RARE factor = 32, matrix = 64×64, FOV = 38.4×38.4 mm², and slice thickness = 3.0 mm. Biodistribution study was conducted by intravenously injecting 100 µL of EP-NPs into 7-week-old healthy mice following the same imaging protocol. An Eppendorf (EP) tube containing PFCE NPs (10.37 mg/mL) in 1.7% agarose served as an internal reference. 2.11. In vivo toxicology evaluation of EP-NPs A toxicity assessment was conducted by intravenously injecting 100 µL of EP-NPs into 7-week-old healthy mice, while the control group received saline. Terminal blood samples were collected via cardiac puncture 30 days post-injection, and serum was promptly transferred to microcentrifuge tubes. Renal and hepatic functions were evaluated by measuring total protein (TP), direct bilirubin (DBILI), indirect bilirubin (IBILI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CRE) and blood urea nitrogen (BUN). Routine blood tests included counts of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT). Organs such as the heart, liver, kidney, spleen, and lung were excised, fixed in 4% paraformaldehyde, dehydrated in graded alcohol, and embedded in paraffin. Tissue sections (5 µm) were stained with hematoxylin and eosin (H&E) and analyzed using an OLYMPUS IX71 microscope (Olympus, Japan). 2.12. Statistical analysis Data analysis was conducted using Prism 8 Software (GraphPad Software, La Jolla, CA). Results are presented as mean ± standard deviation. Group comparisons were evaluated using Student’s t-test, while differences among multiple groups were analyzed by ANOVA. Error bars represent the standard error of the mean (SEM) unless specified. A P-value < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of EP-NPs Dynamic laser scattering (DLS) showed that the hydrodynamic size of EP-NPs was 111.6 ± 8.2 nm (Fig. 1 a). The transmission electron microscopy (TEM) image demonstrated that EP-NPs have regular shape morphology (Fig. 1 b). Zeta potentials of EP-NPs and non-targeted PFCE NPs were − 20.50 ± 1.25 mV and − 4.09 ± 0.83 mV, respectively (Fig. 1 c). As shown in Fig. 1 f, the presence of the exosome characteristic membrane proteins CD63 and CD9 confirming that the exosomal membrane successfully encapsulated PFCE NPs (Fig. 1 d). Also, a single resonance peak indicated that the PFCE-based NPs possessed optimal characteristics for imaging (Fig. 1 e). The MRI of phantoms with EP-NPs showed a highly linear (R2 = 0.9769) correlation of signal-to-noise ratio (SNR) at a range of PFCE concentrations from 4.8 to 19.2 mmol/L (Fig. 2 a-c). This linear relationship between the phantom concentration and SNR indicated that the 19F MRI signal intensity was dependent only on the concentration of PFCE, which is beneficial for the quantification in vivo. 3.2. In vitro cellular uptake study In order to evaluate the influence of exosome membrane coating on the cellular uptake, BT-549 and NCI-H446 cells were chosen as cell models. After 4 h of incubation with EP-NPs or non-targeted PFCE NPs, cells were assessed for nanoparticle uptake using confocal laser scanning microscopy (CLSM). As shown in Fig. 3 a, negligible fluorescence of non-targeted PFCE NPs was observed in both cell models. However, the CLSM images showed that the EP-NPs group had significantly stronger fluorescence signals (Fig. 3 b). The quantitative results further confirming the excellent tumor-targeting capability of exosome membrane (Fig. 3 c). 3.3. In vivo liver metastasis targeted 19 F MRI A BT-549 mouse model was used to evaluate the liver metastasis targeting efficacy of EP-NPs. On the 30th day after the tumor implantation, the mice were divided into two groups, and the 19 F MRI was used to observe the tumor progression (Fig. 4 a). As shown in Fig. 4 b, despite background 19 F signal of liver tissue, small liver metastasis could still be detected on the 7th day after injection of EP-NPs. Meanwhile, liver paraffin section was photographed and analyzed by H&E staining, indicating that there was apartment area of tumor, which were consistent with the 19 F MRI results (Fig. 4 c). In contrast, in the group of non-targeted PFCE NPs, no significant liver metastasis was detected by 19 F MRI throughout the whole observation period (Fig. 4 d), in spite of obvious abnormal 1 H signal in the liver on the 7th day after injection (Fig. 4 e). 3.4. Biodistribution of nanoparticles given intravenous Tissue biodistribution study of EP-NPs was studied in mice using a using a 9.4T MR scanner. As shown in Fig. 5 a, EP-NPs were distributed in the liver and spleen after intravenous injection, but there was no significant 19 F signal in the kidneys, lungs, or intestines throughout the whole observation period. Moreover, no significant decrease in 19 F signal was observed, demonstrates that EP-NPs are characterized by a relatively long circulation time in vivo . 3.5. Toxicity study of EP-NPs No apparent toxicity gainst BT-549 and NCI-H446 cells was detected by MTT assays (Fig. 5 b). Additionally, the serum biochemistry of mice was tested 30 days after injection of EP-NPs. The routine blood (Fig. 5 c-f) of the control group and the EP-NPs treated group showed no obvious different performance. The levels of liver and kidney function markers were within a normal range (Fig. 6 a-f), which confirmed negligible hepatic or renal toxicity of EP-NPs. Moreover, detailed histological analyses of liver, spleen, lung, heart, and kidney tissues showed no observable tissue damage (Fig. 6 g). 4. Discussion The development of EP-NPs, an exosome-biomimetic nanoplatform, represents a significant advancement in the molecular imaging of hepatic metastases. This study addresses the critical limitations of conventional imaging modalities, such as proton-weighted MRI, which suffers from physiological interference due to endogenous water protons and carbohydrate derivatives. By leveraging the unique advantages of 19 F MRI, notably its negligible background signal and quantitative imaging capabilities, combined with the innate targeting properties of exosomes, EP-NPs demonstrate enhanced specificity and sensitivity for LMs detection (Chapelin, Capitini, and Ahrens 2018 ; Euan Martínez et al. 2025). A key innovation of this work lies in the integration of exosomal membranes with perfluorocarbon nanoparticles. Exosomes, as natural intercellular communicators, inherently express membrane proteins (e.g., tetraspanins and integrins) that facilitate tumor-specific homing (Jafari et al. 2020). ​ ​ Furthermore​​, their uptake by recipient cells occurs through various mechanisms including receptor-mediated binding and internalization (Kamerkar et al. 2017 ). Based on the traits that exosomes possessed, this approach addresses receptor redundancy, heterogeneity and off-target effects, enables EP-NPs achieved a high-performance targeted and precise 19 F MR imaging of LMs. In addition, it is worth noting that compared to gadolinium-based contrast agents (GBCAs), which have limited circulation half-life within the bloodstream and must be administered for each proton-weighted MRI procedure to facilitate real-time disease assessmen, EP-NPs, integrating the unique properties of exosomes and PFCE, exhibit long blood circulation times and sustained 19 F signal, enabling real-time monitoring of LMs (Weng et al. 2019 ; Li et al. 2020 ; Li et al. 2023 ). These findings not only highlight the ability of EP-NPs for precise LMs imaging, but also underscore the potential for longitudinal tracking of metastatic progression, which are critical unmet need in clinical oncology. The biosafety profile of EP-NPs further supports their clinical potential. Despite the widespread clinical utility of GBCAs, their safety profile remain subjects of ongoing concern. The association between GBCAs and Nephrogenic Systemic Fibrosis (NSF) in patients with severe renal impairment represents one of the most severe complications. NSF, characterized by progressive dermal and systemic fibrosis, has been predominantly linked to linear GBCAs due to their lower thermodynamic stability and higher propensity for gadolinium ion (Gd³⁺) release (Wahsner et al. 2019 ). Although the incidence of NSF has declined with stricter screening protocols and preferential use of macrocyclic agents, residual risks persist in undiagnosed renal dysfunction cases (Wagner, Drel, and Gorin 2016 ). Emerging evidence confirms gadolinium retention in the brain, bones, and other tissues even in patients with normal renal function (Di Gregorio et al. 2018 ; Lancelot 2016 ; Darrah et al. 2009 ). Post-mortem studies demonstrate 4–100 times higher gadolinium levels in the dentate nucleus and globus pallidus of patients with repeated GBCA exposure (Kanda et al. 2015 ). In this study, cytotoxicity assays revealed minimal impact on cell viability, even at high concentrations, while in vivo toxicology evaluations confirmed no significant alterations in hepatic, renal, or hematological parameters. Histopathological analysis of major organs further corroborated the biocompatibility of EP-NPs, addressing concerns about nanoparticle-induced toxicity. Such safety metrics are critical for translational applications, particularly in patients with advanced malignancies. 5. Conculsion In summary, EP-NPs establish a robust framework for precise LMs imaging, combining the strengths of 19 F MRI and exosome-mediated delivery. This platform not only overcomes the limitations of conventional imaging but also opens avenues for theranostic applications. As next steps, validation in larger animal models and early-phase clinical trials will be essential to translate this innovation into routine clinical practice, ultimately improving prognoses for patients with hepatic metastases. Declarations Aknowledgements Not applicable Authorship contributions Conceptualization: ZX; data curation: ZX, HZ and LC; visualization: ZX and DL; writing—original draft: ZX; writing—review & editing: XD and DL. All the authors have read and approved the final manuscript. Funding This work was supported by the Heilongjiang Provincial Health Commission's Scientific Research foundation (20240909040143). Data availability No datasests were generated or analysed during the current study. Ethics approval and consent to participate All procedures were approved by the Medical Ethics Committee of the Fourth Hospital of Harbin Medical University and adhered to the national standard for laboratory animal welfare (GB/T 35892-2018). Competing interest The authors declare no potential conflicts of interest. References Berkova Z, Zacharovova K, Patikova A, Leontovyc I, Hladikova Z, Cerveny D, Tihlarikova E, Nedela V, Girman P, Jirak D, Saudek F (2022) 'Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats'. J Funct Biomater, 13 Chapelin F, Capitini CM, Ahrens ET (2018) Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer. J Immunother Cancer 6:105 Cheng G, Li W, Ha L, Han X, Hao S, Wan Y, Wang Z, Dong F, Zou X, Mao Y, Zheng SY (2018) Self-Assembly of Extracellular Vesicle-like Metal-Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins. J Am Chem Soc 140:7282–7291 Darrah TH, Prutsman-Pfeiffer JJ, Poreda RJ, Ellen Campbell M, Hauschka PV, Hannigan RE (2009) 'Incorporation of excess gadolinium into human bone from medical contrast agents', Metallomics , 1: 479 – 88 Di Gregorio E, Ferrauto G, Furlan C, Lanzardo S, Nuzzi R, Gianolio E, Aime S (2018) The Issue of Gadolinium Retained in Tissues: Insights on the Role of Metal Complex Stability by Comparing Metal Uptake in Murine Tissues Upon the Concomitant Administration of Lanthanum- and Gadolinium-Diethylentriamminopentaacetate. Invest Radiol 53:167–172 Euan Martínez AA, Bergmann AK, Tellkamp F, Schott-Verdugo S, Bouvain P, Steinhausen J, Bahr J, Kmietczyk V, Bencun M, Flögel U, Distler JHW, Krueger M, Völkers M, Czekelius C, Gohlke H, Temme S, Hesse J and J. Schrader. 2025. 'CD63 as novel target for nanoemulsion-based (19)F MRI imaging and drug delivery to activated cardiac fibroblasts'. Theranostics, 15: 1–18 Havlicek D, Panakkal VM, Voska L, Sedlacek O, Jirak D (2024) Self-Assembled Fluorinated Nanoparticles as Sensitive and Biocompatible Theranostic Platforms for (19)F MRI. Macromol Biosci 24:e2300510 Jafari D, Shajari S, Jafari R, Mardi N, Gomari H, Ganji F, Forouzandeh M, Moghadam and A. Samadikuchaksaraei. 2020. 'Designer Exosomes: A New Platform for Biotechnology Therapeutics', BioDrugs , 34: 567 – 86 Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R (2017) Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546:498–503 Kanda T, Fukusato T, Matsuda M, Toyoda K, Oba H, Kotoku J, Haruyama T, Kitajima K, Furui S (2015) Gadolinium-based Contrast Agent Accumulates in the Brain Even in Subjects without Severe Renal Dysfunction: Evaluation of Autopsy Brain Specimens with Inductively Coupled Plasma Mass Spectroscopy. Radiology 276:228–232 Labrijn AF, Janmaat ML, Reichert JM, Pwhi Parren (2019) Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18:585–608 Lancelot E (2016) Revisiting the Pharmacokinetic Profiles of Gadolinium-Based Contrast Agents: Differences in Long-Term Biodistribution and Excretion. Invest Radiol 51:691–700 Li D, Yang J, Xu Z, Li Y, Sun Y, Wang Y, Zou H, Wang K, Yang L, Wu L, Sun X (2023) c-Met-Targeting (19)F MRI Nanoparticles with Ultralong Tumor Retention for Precisely Detecting Small or Ill-Defined Colorectal Liver Metastases. Int J Nanomed 18:2181–2196 Li S, Wu Y, Ding F, Yang J, Li J, Gao X, Zhang C, Feng J (2020) Engineering macrophage-derived exosomes for targeted chemotherapy of triple-negative breast cancer. Nanoscale 12:10854–10862 Lim SM, Pyo KH, Soo RA and B. C. Cho. 2021. 'The promise of bispecific antibodies: Clinical applications and challenges'. Cancer Treat Rev, 99: 102240 Lucchetti D, Colella F, Artemi G, Haque S, Sgambato A, Pellicano R, Fagoonee S (2023) Smart nano-sized extracellular vesicles for cancer therapy: Potential theranostic applications in gastrointestinal tumors. Crit Rev Oncol Hematol 191:104121 Ma Y, Dong S, Li X, Kim BYS, Yang Z, Jiang W (2020) Extracellular Vesicles: An Emerging Nanoplatform for Cancer Therapy. Front Oncol 10:606906 Milette S, Sicklick JK, Lowy AM, Brodt P (2017) Molecular Pathways: Targeting the Microenvironment of Liver Metastases. Clin Cancer Res 23:6390–6399 Patil S, Jirák D, Saudek F, Hájek M, Scheffler K (2011) Positive contrast visualization of SPIO-labeled pancreatic islets using echo-dephased steady-state free precession. Eur Radiol 21:214–220 Renzulli M, Clemente A, Ierardi AM, Pettinari I, Tovoli F, Brocchi S, Peta G, Cappabianca S, Carrafiello G, Golfieri R (2020) 'Imaging of Colorectal Liver Metastases: New Developments and Pending Issues'. Cancers (Basel), 12 Ruiz-Cabello J, Barnett BP, Bottomley PA, Bulte JW (2011) Fluorine (19F) MRS and MRI in biomedicine. NMR Biomed 24:114–129 Shao F, He R, Jin Y, Pang Y, Sun Z, Zhang B, Jin S (2025) 'Regulation and Clinical Application of Exosomes in Venous Thrombosis', Angiology : 33197251319597 Srivastava A, Rathore S, Munshi A, Ramesh R (2022) Organically derived exosomes as carriers of anticancer drugs and imaging agents for cancer treatment. Semin Cancer Biol 86:80–100 Sun Y, Yang J, Li Y, Luo J, Sun J, Li D, Wang Y, Wang K, Yang L, Wu L, Sun X (2022) Single low-dose INC280-loaded theranostic nanoparticles achieve multirooted delivery for MET-targeted primary and liver metastatic NSCLC. Mol Cancer 21:212 Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, Wei J, Nie G (2014) A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 35:2383–2390 Wagner B, Drel V, Gorin Y (2016) Pathophysiology of gadolinium-associated systemic fibrosis. Am J Physiol Ren Physiol 311:F1–f11 Wahsner J, Gale EM, Rodríguez-Rodríguez A, Caravan P (2019) Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem Rev 119:957–1057 Wan L, Pantel K, Kang Y (2013) Tumor metastasis: moving new biological insights into the clinic. Nat Med 19:1450–1464 Weng Q, Hu X, Zheng J, Xia F, Wang N, Liao H, Liu Y, Kim D, Liu J, Li F, He Q, Yang B, Chen C, Hyeon T, Ling D (2019) Toxicological Risk Assessments of Iron Oxide Nanocluster- and Gadolinium-Based T1MRI Contrast Agents in Renal Failure Rats. ACS Nano 13:6801–6812 Wu L, Wen X, Wang X, Wang C, Sun X, Wang K, Zhang H, Williams T, Stacy AJ, Chen J, Schmieder AH, Lanza GM, Shen B (2018) Local Intratracheal Delivery of Perfluorocarbon Nanoparticles to Lung Cancer Demonstrated with Magnetic Resonance Multimodal Imaging. Theranostics 8:563–574 Xu X, Zhang R, Liu F, Ping J, Wen X, Wang H, Wang K, Sun X, Zou H, Shen B, Wu L (2018) (19)F MRI in orthotopic cancer model via intratracheal administration of α(ν)β(3)-targeted perfluorocarbon nanoparticles. Nanomed (Lond) 13:2551–2562 Yang Y, Hong Y, Cho E, Kim GB, Kim IS (2018) Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. J Extracell Vesicles 7:1440131 Zeng H, Guo S, Ren X, Wu Z, Liu S, Yao X (2023) 'Current Strategies for Exosome Cargo Loading and Targeting Delivery', Cells , 12 Zhan Q, Yi K, Qi H, Li S, Li X, Wang Q, Wang Y, Liu C, Qiu M, Yuan X, Zhao J, Hou X, Kang C (2020) 'Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy', Theranostics , 10: 7889 – 905 Zhang C, Yan K, Fu C, Peng H, Hawker CJ, Whittaker AK (2022) Biological Utility of Fluorinated Compounds: from Materials Design to Molecular Imaging, Therapeutics and Environmental Remediation. Chem Rev 122:167–208 Zhang LY, Yang X, Wang SB, Chen H, Pan HY, Hu ZM (2020) Membrane Derived Vesicles as Biomimetic Carriers for Targeted Drug Delivery System. Curr Top Med Chem 20:2472–2492 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2026 Read the published version in Cancer Nanotechnology → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 02 Oct, 2025 Reviews received at journal 28 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 07 Sep, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7556946","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516533303,"identity":"4bd1fd8b-6d96-4aab-a0f3-9806e9cbf8c7","order_by":0,"name":"Zuoyu Xu","email":"","orcid":"","institution":"The Fourth Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zuoyu","middleName":"","lastName":"Xu","suffix":""},{"id":516533304,"identity":"7dd45d04-5800-4d2c-a57a-cf82ba17080a","order_by":1,"name":"Hongyan Zou","email":"","orcid":"","institution":"The Fourth Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Zou","suffix":""},{"id":516533305,"identity":"bdf6042d-504a-494a-9588-1ce092703c7b","order_by":2,"name":"Lixin Cheng","email":"","orcid":"","institution":"The Fourth Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lixin","middleName":"","lastName":"Cheng","suffix":""},{"id":516533306,"identity":"8b7dd49d-4793-4d13-86b3-05853c8a7d86","order_by":3,"name":"Xiaoqiu Dong","email":"","orcid":"","institution":"The Fourth Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqiu","middleName":"","lastName":"Dong","suffix":""},{"id":516533307,"identity":"929b8343-e657-4600-81f8-fcb7c45aa263","order_by":4,"name":"Daoshuang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBACNv7mAwc+/rGpZ2NvPvggoaKGsBY+iWOJD2c2pCXw8RxLNnhw5hhhLXIMOcbGnA2HE+QkcswkH7YwE+EwhgNm0ow70vLYGBLMKhIb2Bj427sT8GthbkiTLjxjUwzUm3YjcYcMg8SZsxsI2XJMegZbGmMbY8OxG4ln2BgMJHIJaUlsk+ZhO8zYxszYVpDYxkyMlmRmY962w4ltbMwg7cRokTjG+HDGmTRjNh42ZomEM8d4CPpFvr//w4EPFTZy8vPff/z4o6JGjr+9F78WDMBDmvJRMApGwSgYBVgBAF5kSylzimh2AAAAAElFTkSuQmCC","orcid":"","institution":"The Fourth Hospital of Harbin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Daoshuang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-09-07 14:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7556946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7556946/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12645-026-00367-0","type":"published","date":"2026-01-29T15:59:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92047517,"identity":"f459f39e-55ad-4f2b-9273-0f552e963bc4","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3847456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/355f6d689b9130d017a524c7.docx"},{"id":92047512,"identity":"770c5a9b-ec63-4726-bfa0-d907d9e0edbe","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6813,"visible":true,"origin":"","legend":"","description":"","filename":"73e2b07726204add95656235076a19f4.json","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/09f6e803ad98918dc28a637a.json"},{"id":92047515,"identity":"d967880f-4afd-44af-89fd-518d28abddde","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100174,"visible":true,"origin":"","legend":"","description":"","filename":"73e2b07726204add95656235076a19f41enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/98b280d01c51f1fea7344c78.xml"},{"id":92047525,"identity":"7a6f7db7-30e2-416b-bd4f-cbe2ca00ae6a","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252130,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/33fbcbc71628eaa12902c02e.jpeg"},{"id":92047528,"identity":"2878c3dc-52c4-4927-9ece-1e2e8deb9a60","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115858,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/8f85e374f0bed470453bdc49.jpeg"},{"id":92063292,"identity":"baa9b58b-30aa-4df4-a2c6-6ad9808856d4","added_by":"auto","created_at":"2025-09-24 08:37:52","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":356914,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/34f8736041b599360418d986.jpeg"},{"id":92047518,"identity":"2ebdc30c-118d-41c1-bfcf-2566d1b6f45e","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":927478,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/aac6f3fd30710dd6c3a809ae.jpeg"},{"id":92047532,"identity":"83315910-9174-4e54-9831-7e7c653bddd9","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":360184,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/d21382a1414acac3f7a73b9f.jpeg"},{"id":92047527,"identity":"195211e6-71b9-458e-b5ba-3e37715caafd","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":802282,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/a7f62a08a2e89040aa8be5b7.jpeg"},{"id":92047520,"identity":"d7640293-3dbf-44dc-b520-9587a86ee3d6","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69206,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/794d6b1e88f8a44dedde5c03.png"},{"id":92047530,"identity":"547f1901-d314-46c1-a218-b847295c0ecf","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32804,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/6b6325b4b50b34d73377ca74.png"},{"id":92063221,"identity":"8b6e1935-794b-4615-ac4f-4f53934f1e10","added_by":"auto","created_at":"2025-09-24 08:37:30","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69995,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/95b89801114223d8f4db891a.png"},{"id":92047531,"identity":"49dba98f-d71b-45b8-bea4-8c427e554499","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":280046,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/4ad8d6e8ba190ca60fa5b32b.png"},{"id":92047529,"identity":"16461cfd-4524-4fd6-80b5-32a58b29fd56","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82448,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/8bf40fc49d4a675749cf9db9.png"},{"id":92047533,"identity":"3925fa01-7d0f-4936-8a78-587602a5d258","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":294903,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/4ee5eed500f5376601e0950f.png"},{"id":92047534,"identity":"6adbc058-0f26-489f-b08c-49e0c4f4f261","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97710,"visible":true,"origin":"","legend":"","description":"","filename":"73e2b07726204add95656235076a19f41structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/876bd9dbb4ede84d5ed155cf.xml"},{"id":92063284,"identity":"0be0f412-2624-4e10-a0ba-60146fed2ee4","added_by":"auto","created_at":"2025-09-24 08:37:49","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104529,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/239ac2eea71ea2734a3e1d16.html"},{"id":92047514,"identity":"ad7c6e83-fedf-4851-9319-e30eb6b428a5","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of EP-NPs. \u003c/strong\u003e(a) Average hydrodynamic size of EP-NPs characterized by dynamic light scattering (DLS). (b) Transmission electron microscopy (TEM) image of EP-NPs; Scale bar is 50 µm. (c) The zeta potential of EP-NPs and non-targeted PFCE NPs. (d) Western blot analysis of the commonly used exosome markers CD9 and CD63 in EP-NPs and PFCE NPs. (e) \u003csup\u003e19\u003c/sup\u003eF NMR spectrum of EP-NPs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/bf1fb49359765df434c42955.png"},{"id":92047513,"identity":"10a62368-3012-4c36-bdae-dfad5ae703d8","added_by":"auto","created_at":"2025-09-24 05:07:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of EP-NPs. \u003c/strong\u003e(a) Color-scaled \u003csup\u003e19\u003c/sup\u003eF-MR image of EP-NPs in 1.7% agarose solution with various \u003csup\u003e19\u003c/sup\u003eF concentrations, where 1=4.8, 2=7.2, 3=9.6, 4=14.4, 5=19.2 (mmol/L). (b) The corresponding \u003csup\u003e1\u003c/sup\u003eH MR image. (c) The concentrations of PFCE versus signal-to-noise ratio (SNR) are plotted with R2=0.9769.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/bd662faed9a0e3cbdcd23c53.png"},{"id":92047526,"identity":"0d4e5d82-cc09-4c22-9061-57ffdf81c0c5","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":692728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of EP-NPs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) CLSM images of BT-549 and NCI-H446 cells in PFCE NPs group. Blue fluorescence indicates nuclei stained with DAPI; red fluorescence represents rhodamine-labeled PFCE NPs; Scale bar is 100 µm. (b) CLSM images of BT-549 and NCI-H446 cells in EP-NPs group. Blue fluorescence indicates nuclei stained with DAPI; green fluorescence represents PKH67-labeled EP-NPs; Scale bar is 100 µm. (c) Mean fluorescence intensity (MFI) quantification of BT-549 and NCI-H446 cells in each group, ** P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/815d12e2fd597ecac1d5e753.png"},{"id":92047523,"identity":"6adb6105-1d5e-4074-bc9f-df9614abbfb0","added_by":"auto","created_at":"2025-09-24 05:07:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":887781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e liver metastasis targeted \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eF MRI.\u003c/strong\u003e (a) Schematic illustration showing the protocol of imaging assay. (b) \u003csup\u003e19\u003c/sup\u003eF MR images of the liver metastasis murine model at different time points in EP-NPs group. Black arrow indicates the metastasis site. (c) H\u0026amp;E staining of liver with metastasis in murine model with EP-NPs administration; Scale bar is 100 µm. (d) \u003csup\u003e19\u003c/sup\u003eF MR images of the liver metastasis murine model at different time points in PFCE NPs group. Black arrow indicates the metastasis site. (e) The corresponding \u003csup\u003e1\u003c/sup\u003eH MR image of the liver metastasis murine model at 7th day in PFCE NPs group. White arrow indicates the metastasis site.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/03f58b28989a2d3d9c0b05d2.png"},{"id":92062908,"identity":"631a66df-d521-495a-b86d-d0dc108a75e9","added_by":"auto","created_at":"2025-09-24 08:35:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":690364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiodistribution and toxicity study of EP-NPs.\u003c/strong\u003e (a) Superimposed \u003csup\u003e1\u003c/sup\u003eH (gray) and \u003csup\u003e19\u003c/sup\u003eF (red) images of healthy mice with EP-NPs administration at different time points. (b) Viability of BT-549 and NCI-H446 cells after incubation with EP-NPs at different concentrations for 24 h. The results are the average of three replicates ± standard deviation. (c-f) Routine blood biochemical data of mice intravenously injected with EP-NPs: (c) white blood cell count (WBC), (d) red blood cell count (RBC), (e) platelet count (PLT), (f) hemoglobin (HGB).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/ac65a7102489ff62da60b925.png"},{"id":92063012,"identity":"9dfd1dfd-332b-4a65-9108-391a83a6105d","added_by":"auto","created_at":"2025-09-24 08:36:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":619954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eToxicity study of EP-NPs.\u003c/strong\u003e (a-f) Indicators of liver and kidney function of mice intravenously injected with EP-NPs: (a) total protein (TP), (b) alanine aminotransferase (ALT), (c) aspartate aminotransferase (AST), (d) creatinine (CRE), (e) blood urea nitrogen (BUN) and (f) hypersensitive C-reactive protein (CRP). (g) H\u0026amp;E staining in major organs (heart, liver, spleen, lung and kidney) of both groups 30 days after injection; Scale bar is 200 µm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/81c0886e8a0d37527e9bcaf6.png"},{"id":101691273,"identity":"6a31df28-6f6e-4768-8225-eff44f71d617","added_by":"auto","created_at":"2026-02-02 16:13:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4142075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7556946/v1/662e2280-3cd9-42fe-b15d-b4423ec7629d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosome-Biomimetic Nanoprobes Achieve Ultrasensitive Detection and Real-Time Monitoring of Liver Metastasis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHepatic metastasis persists as the principal contributor to cancer-related mortality worldwide (Wan, Pantel, and Kang \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The unique hepatic microenvironment, characterized by extensive vascularization and sinusoidal tissue architecture, provides an optimal niche for circulating tumor cell colonization, resulting in frequent liver metastasis (LM) across multiple malignancies (Milette et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Detection of LMs constitutes a critical determinant for therapeutic outcomes. In clinical practice for detecting hepatic metastases, current imaging modalities primarily include computed tomography (CT), ultrasonography, and magnetic resonance imaging (MRI). Notably, gadolinium-based MRI demonstrates superior detection sensitivity among these techniques (Renzulli et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, conventional proton-weighted MRI encounters inherent limitations including substantial physiological interference from endogenous water protons and carbohydrate derivatives (Berkova et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patil et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo circumvent these constraints, fluorine-19 (\u003csup\u003e19\u003c/sup\u003eF) MRI has emerged as an innovative molecular imaging strategy (Zhang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Havlicek et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This modality capitalizes on the negligible endogenous fluorine background in biological systems, enabling quantitative hotspot imaging of exogenously administered fluorinated probes with enhanced signal specificity (Ruiz-Cabello et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Perfluorocarbon nanoparticles (PFC NPs) functionalized with targeting ligands have shown promise in tumor-specific \u003csup\u003e19\u003c/sup\u003eF MRI applications (Xu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, tumor heterogeneity and receptor expression variability across cancer subtypes constrained the clinical translation of these imaging agents that targeting only a single site (Labrijn et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lim et al. 2021).\u003c/p\u003e\u003cp\u003eRecent advances in extracellular vesicle engineering have highlighted exosomes as promising theranostic vectors (Zeng et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lucchetti et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Exosomes refer to a group of extracellular vesicles naturally released from mammalian cells with a diameter of 30\u0026ndash;150 nm (Shao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Exosomes, functioning as nanoscale signaling vesicles, carry diverse nucleic acids and proteins, facilitating intercellular communication. Their membranes are embedded with various functional proteins that perform specific roles. What is more important, exosomes possess inherent cell-targeting capabilities due to the presence of specific membrane proteins, including tetraspanins and integrins (Yang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, the ability of exosomes to shuttle molecules has facilitated their use as alternative vehicles for delivering molecular imaging agents within their lumen, transferring them to cells at distant sites in the body (Srivastava et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All these distinctive characteristics position exosomes as exceptional nanoplatforms for imaging applications.\u003c/p\u003e\u003cp\u003eIn this investigation, we engineered a novel exosome-biomimetic nanoprobe named EP-NPs, using an exosomal membrane coated PFC NPs to achieve efficient and precise imaging of LMs. Systematic evaluation of tumor-targeting efficiency, biosafety profiles, and imaging performance revealed that the exosome-coated nanoplatform possess excellent tumor accumulation efficacy, high biocompatibility and sustained \u003csup\u003e19\u003c/sup\u003eF signal persistence.This nanoplatform generates a strategy for real-time monitoring and early detection of LMs.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eCell culture media (RPMI-1640, Leibovitz\u0026rsquo;s L-15, DMEM), fetal bovine serum (FBS), and Trypsin-EDTA (0.25%) were supplied by Thermo Fisher Scientific Inc. (SHH, CHN). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent and dimethyl sulfoxide (DMSO) were procured from Sigma-Aldrich (SHH, CHN) and Tansoolereagent (SHH, CHN), respectively. Human cell lines, BT-549 (breast carcinoma), NCI-H446 (lung carcinoma), and HEK-293T (embryonic kidney)were acquired from Procell Life Science \u0026amp; Technology Co., Ltd. (HB, CHN).\u003c/p\u003e\u003cp\u003ePerfluoro-15-crown-5-ether (PFCE, C\u003csub\u003e10\u003c/sub\u003eF\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) was sourced from Exfluor Research Corporation (TX, USA), while lecithin and cholesterol (Avanti Polar Lipids, AL, USA) and glycerin (Aldrich, WI, USA) constituted the lipid components. Ultra-high-purity argon (99.99%) was utilized for material storage. Dialysis membranes (20 kDa MWCO, Spectrum Medical Industries, CA, USA) and analytical-grade chemicals were employed without further purification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell culture\u003c/h2\u003e\u003cp\u003eBT-549 and NCI-H446 cells were cultured in RPMI-1640 (supplemented with 10% FBS), HEK-293T was cultured in DMEM (supplemented with 10% FBS), all cells were cultured in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃ and passed around 80% confluence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Preparation of PFC NPs\u003c/h2\u003e\u003cp\u003ePFC NPs were prepared as per prior protocols (Wu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The emulsion comprised 20% (v/v) PFCE, 2% (w/v) surfactant blend (85 mol% lecithin, 2.9 mol% phosphatidylglycerol, 12 mol% cholesterol), 2.5% (w/v) glycerin, and water. Lipid films were formed by dissolving components in methanol-chloroform, evaporating solvents via rotary evaporation (45\u0026deg;C), and vacuum drying (24 h). Hydration with purified water, followed by vortexing (2 min) and probe sonication (2 min), yielded nanoparticle suspensions. Final emulsions were dialyzed (20 kDa MWCO cellulosic membrane), argon-purged, and stored at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of exosome membrane coated PFC NPs\u003c/h2\u003e\u003cp\u003eExosomes were harvested by collecting the supernatant from HEK-293T cell cultures grown in T225 flasks at 80% confluency, followed by isolation using differential centrifugation as described in previous studies (Zhan et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All procedures were performed at 4\u0026deg;C. Initially, the exosome-containing supernatant was centrifuged at 3000g for 30 min to eliminate dead cells and debris. Subsequently, the supernatant underwent centrifugation at 10,000g for 45 min to remove larger vesicles. The clarified supernatant was then filtered through a 0.22 \u0026micro;m membrane (Millipore, USA). Exosomes were pelleted via ultracentrifugation at 100,000g for 90 min, resuspended in PBS, sonicated, and passed through a 200 nm microporous membrane to achieve a homogeneous suspension. The contents of exosomes were removed by hypotonic treatment and ultracentrifugation (Cheng et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For exosome membrane-coated perfluorocarbon nanoparticles, empty exosome vesicles were combined with PFCE NPs and extruded 10 times through a 100 nm polycarbonate membrane using an Avanti mini extruder, yielding exosome-coated nanoparticles (EP-NPs).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Characterization of nanoparticles\u003c/h2\u003e\u003cp\u003eThe hydrodynamic diameter,and zeta potential (ζ) of EP-NPs were analyzed using a Malvern Nano ZS Zetasizer (Malvern Instruments Ltd, UK) via dynamic light scattering (DLS). All measurements were repeated three times for consistency.The morphology of nanoparticles was observed with a Talos F200s transmission electron microscope (FEI, ThermoFisher, Waltham, MA) operating at 200 kV. Elemental mapping images were also obtained by using transmission electron microscopy (TEM).\u003c/p\u003e\u003cp\u003e\u003csup\u003e19\u003c/sup\u003eF NMR characterization was performed using a 9.4T Bruker Avance III 400 MHz spectrometer. Spectra were acquired using a \u0026ldquo;single-pulse\u0026rdquo; sequence (Bruker\u0026ldquo;zg\u0026rdquo;sequence), with a D\u003csub\u003e2\u003c/sub\u003eO-filled capillary tube for field locking. Measurements were taken at -75.4 ppm, with a 50 ppm sweep width, 8 scans, and a 25-second interscan delay. Data processing was conducted by using Mestrenova 10.0.2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Phantom experiments\u003c/h2\u003e\u003cp\u003eEP-NPs were dispersed in 1.7% agarose at PFCE concentrations of 4.8, 7.2, 9.6, 14.4, and 19.2 mmol/L and loaded into tubes. \u003csup\u003e19\u003c/sup\u003eF MR imaging was performed using a 9.4T small animal scanner (BioSpec 94/20 USR, Bruker, Germany) with a multi-slice multi-echo (MSME) sequence, featuring 24 echo times (TE) from 8 to 192 msec at 8-msec intervals. Imaging parameters included: repetition time (TR), 3000 msec; matrix size, 64\u0026times;64; field of view (FOV), 38.4\u0026times;38.4 mm; and slice thickness, 1 mm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Cellular imaging experiment of EP-NPs\u003c/h2\u003e\u003cp\u003eBT-549 and NCI-H446 cells were plated in confocal microscopy dishes at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per dish. Following a 24-hour incubation, the cells were treated with either EP-NPs (40 \u0026micro;L/mL culture media) or non-targeted PFCE NPs (40 \u0026micro;L/mL culture media). The PFCE nanoparticles in EP-NPs and non-targeted PFCE NPs were labeled with PKH67 and Rhodamine, respectively. After a 4-hour incubation at 37\u0026deg;C, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Subsequent to three PBS washes, the nuclei were stained with 4\u0026rsquo;6-diamidino-2-phenylindole (DAPI). The dishes were then washed three more times, and cellular uptake was observed by confocal laser scanning microscopy (Nikon, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Cytotoxicity assay of EP-NPs\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of EP-NPs was assessed using the MTT assay. BT-549 and NCI-H446 cells were seeded in 96-well plates at 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated for 24 hours. Cells were then treated with varying concentrations of EP-NPs (PFCE concentrations were 4.8, 7.2, 9.6, 14.4, and 19.2 mmol/L) in a final volume of 200 \u0026micro;L/well and incubated overnight at 37\u0026deg;C. Following this, 20 \u0026micro;L of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 4 hours. The medium was then replaced with 150 \u0026micro;L of DMSO, and absorbance at 490 nm was measured using a plate reader to determine cell viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Animal models\u003c/h2\u003e\u003cp\u003eFemale BALB/c nude mice, aged 6\u0026ndash;8 weeks, were sourced from Vital River Laboratory Animal Technology Co., Ltd. (BJ, CHN) and housed in specific pathogen-free conditions with unrestricted access to food and water. For the liver metastasis model, BT-549 cells were cultured and suspended in sterile PBS. Mice were anesthetized with isoflurane and positioned supine. A small left flank incision was made to expose the spleen using a retractor. A suspension of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e BT-549 cells in 50 \u0026micro;L PBS was injected into the spleen parenchyma. The spleen was then returned to the abdominal cavity, and the incision was stitched up with a 5\u0026thinsp;\u0026minus;\u0026thinsp;0 absorbable suture\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. \u003cem\u003eIn vivo\u003c/em\u003e liver metastasis targeting and biodistribution studies.\u003c/h2\u003e\u003cp\u003eAt the predetermined time points, mice were anesthetized with isoflurane and imaged using a 9.4T MR scanner (BioSpec 94/20 USR, Bruker) with a \u003csup\u003e1\u003c/sup\u003eH/\u003csup\u003e19\u003c/sup\u003eF dual-tune volume coil. For \u003cem\u003ein vivo\u003c/em\u003e tumor targeting study, \u003csup\u003e1\u003c/sup\u003eH (T2-weighted) and \u003csup\u003e19\u003c/sup\u003eF images were obtained from mice in the EP-NPs group (100 \u0026micro;L/mouse) and the non-targeted PFCE NPs group (100 \u0026micro;L/mouse) before and after intravenous injection. \u003csup\u003e1\u003c/sup\u003eH imaging utilized a rapid acquisition with relaxation enhancement (RARE) sequence with the following scan parameters: TR/TE\u0026thinsp;=\u0026thinsp;3000/40 msec, NA\u0026thinsp;=\u0026thinsp;2, RARE factor\u0026thinsp;=\u0026thinsp;10, matrix\u0026thinsp;=\u0026thinsp;256\u0026times;256, FOV\u0026thinsp;=\u0026thinsp;38.4\u0026times;38.4 mm\u0026sup2;, and slice thickness\u0026thinsp;=\u0026thinsp;1.0 mm. The \u003csup\u003e19\u003c/sup\u003eF RARE sequence, aligned with the proton images, was acquired with TR/TE\u0026thinsp;=\u0026thinsp;2000/100 msec, NA\u0026thinsp;=\u0026thinsp;128, RARE factor\u0026thinsp;=\u0026thinsp;32, matrix\u0026thinsp;=\u0026thinsp;64\u0026times;64, FOV\u0026thinsp;=\u0026thinsp;38.4\u0026times;38.4 mm\u0026sup2;, and slice thickness\u0026thinsp;=\u0026thinsp;3.0 mm. Biodistribution study was conducted by intravenously injecting 100 \u0026micro;L of EP-NPs into 7-week-old healthy mice following the same imaging protocol. An Eppendorf (EP) tube containing PFCE NPs (10.37 mg/mL) in 1.7% agarose served as an internal reference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. \u003cem\u003eIn vivo\u003c/em\u003e toxicology evaluation of EP-NPs\u003c/h2\u003e\u003cp\u003eA toxicity assessment was conducted by intravenously injecting 100 \u0026micro;L of EP-NPs into 7-week-old healthy mice, while the control group received saline. Terminal blood samples were collected via cardiac puncture 30 days post-injection, and serum was promptly transferred to microcentrifuge tubes. Renal and hepatic functions were evaluated by measuring total protein (TP), direct bilirubin (DBILI), indirect bilirubin (IBILI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CRE) and blood urea nitrogen (BUN). Routine blood tests included counts of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT). Organs such as the heart, liver, kidney, spleen, and lung were excised, fixed in 4% paraformaldehyde, dehydrated in graded alcohol, and embedded in paraffin. Tissue sections (5 \u0026micro;m) were stained with hematoxylin and eosin (H\u0026amp;E) and analyzed using an OLYMPUS IX71 microscope (Olympus, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e\u003cp\u003eData analysis was conducted using Prism 8 Software (GraphPad Software, La Jolla, CA). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Group comparisons were evaluated using Student\u0026rsquo;s t-test, while differences among multiple groups were analyzed by ANOVA. Error bars represent the standard error of the mean (SEM) unless specified. A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of EP-NPs\u003c/h2\u003e\u003cp\u003eDynamic laser scattering (DLS) showed that the hydrodynamic size of EP-NPs was 111.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The transmission electron microscopy (TEM) image demonstrated that EP-NPs have regular shape morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Zeta potentials of EP-NPs and non-targeted PFCE NPs were \u0026minus;\u0026thinsp;20.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 mV and \u0026minus;\u0026thinsp;4.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 mV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, the presence of the exosome characteristic membrane proteins CD63 and CD9 confirming that the exosomal membrane successfully encapsulated PFCE NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Also, a single resonance peak indicated that the PFCE-based NPs possessed optimal characteristics for imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe MRI of phantoms with EP-NPs showed a highly linear (R2\u0026thinsp;=\u0026thinsp;0.9769) correlation of signal-to-noise ratio (SNR) at a range of PFCE concentrations from 4.8 to 19.2 mmol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). This linear relationship between the phantom concentration and SNR indicated that the 19F MRI signal intensity was dependent only on the concentration of PFCE, which is beneficial for the quantification in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. \u003cem\u003eIn vitro\u003c/em\u003e cellular uptake study\u003c/h2\u003e\u003cp\u003eIn order to evaluate the influence of exosome membrane coating on the cellular uptake, BT-549 and NCI-H446 cells were chosen as cell models. After 4 h of incubation with EP-NPs or non-targeted PFCE NPs, cells were assessed for nanoparticle uptake using confocal laser scanning microscopy (CLSM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, negligible fluorescence of non-targeted PFCE NPs was observed in both cell models. However, the CLSM images showed that the EP-NPs group had significantly stronger fluorescence signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The quantitative results further confirming the excellent tumor-targeting capability of exosome membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. \u003cem\u003eIn vivo\u003c/em\u003e liver metastasis targeted \u003csup\u003e19\u003c/sup\u003eF MRI\u003c/h2\u003e\u003cp\u003eA BT-549 mouse model was used to evaluate the liver metastasis targeting efficacy of EP-NPs. On the 30th day after the tumor implantation, the mice were divided into two groups, and the \u003csup\u003e19\u003c/sup\u003eF MRI was used to observe the tumor progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, despite background \u003csup\u003e19\u003c/sup\u003eF signal of liver tissue, small liver metastasis could still be detected on the 7th day after injection of EP-NPs. Meanwhile, liver paraffin section was photographed and analyzed by H\u0026amp;E staining, indicating that there was apartment area of tumor, which were consistent with the \u003csup\u003e19\u003c/sup\u003eF MRI results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In contrast, in the group of non-targeted PFCE NPs, no significant liver metastasis was detected by \u003csup\u003e19\u003c/sup\u003eF MRI throughout the whole observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), in spite of obvious abnormal \u003csup\u003e1\u003c/sup\u003eH signal in the liver on the 7th day after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Biodistribution of nanoparticles given intravenous\u003c/h2\u003e\u003cp\u003eTissue biodistribution study of EP-NPs was studied in mice using a using a 9.4T MR scanner. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, EP-NPs were distributed in the liver and spleen after intravenous injection, but there was no significant \u003csup\u003e19\u003c/sup\u003eF signal in the kidneys, lungs, or intestines throughout the whole observation period. Moreover, no significant decrease in \u003csup\u003e19\u003c/sup\u003eF signal was observed, demonstrates that EP-NPs are characterized by a relatively long circulation time \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Toxicity study of EP-NPs\u003c/h2\u003e\u003cp\u003eNo apparent toxicity gainst BT-549 and NCI-H446 cells was detected by MTT assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Additionally, the serum biochemistry of mice was tested 30 days after injection of EP-NPs. The routine blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-f) of the control group and the EP-NPs treated group showed no obvious different performance. The levels of liver and kidney function markers were within a normal range (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-f), which confirmed negligible hepatic or renal toxicity of EP-NPs. Moreover, detailed histological analyses of liver, spleen, lung, heart, and kidney tissues showed no observable tissue damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe development of EP-NPs, an exosome-biomimetic nanoplatform, represents a significant advancement in the molecular imaging of hepatic metastases. This study addresses the critical limitations of conventional imaging modalities, such as proton-weighted MRI, which suffers from physiological interference due to endogenous water protons and carbohydrate derivatives. By leveraging the unique advantages of \u003csup\u003e19\u003c/sup\u003eF MRI, notably its negligible background signal and quantitative imaging capabilities, combined with the innate targeting properties of exosomes, EP-NPs demonstrate enhanced specificity and sensitivity for LMs detection (Chapelin, Capitini, and Ahrens \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Euan Mart\u0026iacute;nez et al. 2025).\u003c/p\u003e\u003cp\u003eA key innovation of this work lies in the integration of exosomal membranes with perfluorocarbon nanoparticles. Exosomes, as natural intercellular communicators, inherently express membrane proteins (e.g., tetraspanins and integrins) that facilitate tumor-specific homing (Jafari et al. 2020). ​\u003cb\u003e​\u003c/b\u003eFurthermore​​, their uptake by recipient cells occurs through various mechanisms including receptor-mediated binding and internalization (Kamerkar et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Based on the traits that exosomes possessed, this approach addresses receptor redundancy, heterogeneity and off-target effects, enables EP-NPs achieved a high-performance targeted and precise \u003csup\u003e19\u003c/sup\u003eF MR imaging of LMs. In addition, it is worth noting that compared to gadolinium-based contrast agents (GBCAs), which have limited circulation half-life within the bloodstream and must be administered for each proton-weighted MRI procedure to facilitate real-time disease assessmen, EP-NPs, integrating the unique properties of exosomes and PFCE, exhibit long blood circulation times and sustained \u003csup\u003e19\u003c/sup\u003eF signal, enabling real-time monitoring of LMs (Weng et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings not only highlight the ability of EP-NPs for precise LMs imaging, but also underscore the potential for longitudinal tracking of metastatic progression, which are critical unmet need in clinical oncology.\u003c/p\u003e\u003cp\u003eThe biosafety profile of EP-NPs further supports their clinical potential. Despite the widespread clinical utility of GBCAs, their safety profile remain subjects of ongoing concern. The association between GBCAs and Nephrogenic Systemic Fibrosis (NSF) in patients with severe renal impairment represents one of the most severe complications. NSF, characterized by progressive dermal and systemic fibrosis, has been predominantly linked to linear GBCAs due to their lower thermodynamic stability and higher propensity for gadolinium ion (Gd\u0026sup3;⁺) release (Wahsner et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although the incidence of NSF has declined with stricter screening protocols and preferential use of macrocyclic agents, residual risks persist in undiagnosed renal dysfunction cases (Wagner, Drel, and Gorin \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Emerging evidence confirms gadolinium retention in the brain, bones, and other tissues even in patients with normal renal function (Di Gregorio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lancelot \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Darrah et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Post-mortem studies demonstrate 4\u0026ndash;100 times higher gadolinium levels in the dentate nucleus and globus pallidus of patients with repeated GBCA exposure (Kanda et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, cytotoxicity assays revealed minimal impact on cell viability, even at high concentrations, while \u003cem\u003ein vivo\u003c/em\u003e toxicology evaluations confirmed no significant alterations in hepatic, renal, or hematological parameters. Histopathological analysis of major organs further corroborated the biocompatibility of EP-NPs, addressing concerns about nanoparticle-induced toxicity. Such safety metrics are critical for translational applications, particularly in patients with advanced malignancies.\u003c/p\u003e"},{"header":"5. Conculsion","content":"\u003cp\u003eIn summary, EP-NPs establish a robust framework for precise LMs imaging, combining the strengths of \u003csup\u003e19\u003c/sup\u003eF MRI and exosome-mediated delivery. This platform not only overcomes the limitations of conventional imaging but also opens avenues for theranostic applications. As next steps, validation in larger animal models and early-phase clinical trials will be essential to translate this innovation into routine clinical practice, ultimately improving prognoses for patients with hepatic metastases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: ZX; data curation: ZX, HZ and LC; visualization: ZX and DL; writing\u0026mdash;original draft: ZX; writing\u0026mdash;review \u0026amp; editing: XD and DL. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Heilongjiang Provincial Health Commission\u0026apos;s Scientific Research foundation (20240909040143).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasests were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Medical Ethics Committee of the Fourth Hospital of Harbin Medical University and adhered to the national standard for laboratory animal welfare (GB/T 35892-2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBerkova Z, Zacharovova K, Patikova A, Leontovyc I, Hladikova Z, Cerveny D, Tihlarikova E, Nedela V, Girman P, Jirak D, Saudek F (2022) 'Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats'. J Funct Biomater, 13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapelin F, Capitini CM, Ahrens ET (2018) Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer. J Immunother Cancer 6:105\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng G, Li W, Ha L, Han X, Hao S, Wan Y, Wang Z, Dong F, Zou X, Mao Y, Zheng SY (2018) Self-Assembly of Extracellular Vesicle-like Metal-Organic Framework Nanoparticles for Protection and Intracellular Delivery of Biofunctional Proteins. J Am Chem Soc 140:7282\u0026ndash;7291\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDarrah TH, Prutsman-Pfeiffer JJ, Poreda RJ, Ellen Campbell M, Hauschka PV, Hannigan RE (2009) 'Incorporation of excess gadolinium into human bone from medical contrast agents', \u003cem\u003eMetallomics\u003c/em\u003e, 1: 479\u0026thinsp;\u0026ndash;\u0026thinsp;88\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Gregorio E, Ferrauto G, Furlan C, Lanzardo S, Nuzzi R, Gianolio E, Aime S (2018) The Issue of Gadolinium Retained in Tissues: Insights on the Role of Metal Complex Stability by Comparing Metal Uptake in Murine Tissues Upon the Concomitant Administration of Lanthanum- and Gadolinium-Diethylentriamminopentaacetate. Invest Radiol 53:167\u0026ndash;172\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEuan Mart\u0026iacute;nez AA, Bergmann AK, Tellkamp F, Schott-Verdugo S, Bouvain P, Steinhausen J, Bahr J, Kmietczyk V, Bencun M, Fl\u0026ouml;gel U, Distler JHW, Krueger M, V\u0026ouml;lkers M, Czekelius C, Gohlke H, Temme S, Hesse J and J. Schrader. 2025. 'CD63 as novel target for nanoemulsion-based (19)F MRI imaging and drug delivery to activated cardiac fibroblasts'. Theranostics, 15: 1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHavlicek D, Panakkal VM, Voska L, Sedlacek O, Jirak D (2024) Self-Assembled Fluorinated Nanoparticles as Sensitive and Biocompatible Theranostic Platforms for (19)F MRI. Macromol Biosci 24:e2300510\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJafari D, Shajari S, Jafari R, Mardi N, Gomari H, Ganji F, Forouzandeh M, Moghadam and A. Samadikuchaksaraei. 2020. 'Designer Exosomes: A New Platform for Biotechnology Therapeutics', \u003cem\u003eBioDrugs\u003c/em\u003e, 34: 567\u0026thinsp;\u0026ndash;\u0026thinsp;86\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, Lee JJ, Kalluri R (2017) Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546:498\u0026ndash;503\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanda T, Fukusato T, Matsuda M, Toyoda K, Oba H, Kotoku J, Haruyama T, Kitajima K, Furui S (2015) Gadolinium-based Contrast Agent Accumulates in the Brain Even in Subjects without Severe Renal Dysfunction: Evaluation of Autopsy Brain Specimens with Inductively Coupled Plasma Mass Spectroscopy. Radiology 276:228\u0026ndash;232\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLabrijn AF, Janmaat ML, Reichert JM, Pwhi Parren (2019) Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov 18:585\u0026ndash;608\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLancelot E (2016) Revisiting the Pharmacokinetic Profiles of Gadolinium-Based Contrast Agents: Differences in Long-Term Biodistribution and Excretion. Invest Radiol 51:691\u0026ndash;700\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi D, Yang J, Xu Z, Li Y, Sun Y, Wang Y, Zou H, Wang K, Yang L, Wu L, Sun X (2023) c-Met-Targeting (19)F MRI Nanoparticles with Ultralong Tumor Retention for Precisely Detecting Small or Ill-Defined Colorectal Liver Metastases. Int J Nanomed 18:2181\u0026ndash;2196\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, Wu Y, Ding F, Yang J, Li J, Gao X, Zhang C, Feng J (2020) Engineering macrophage-derived exosomes for targeted chemotherapy of triple-negative breast cancer. Nanoscale 12:10854\u0026ndash;10862\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim SM, Pyo KH, Soo RA and B. C. Cho. 2021. 'The promise of bispecific antibodies: Clinical applications and challenges'. Cancer Treat Rev, 99: 102240\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLucchetti D, Colella F, Artemi G, Haque S, Sgambato A, Pellicano R, Fagoonee S (2023) Smart nano-sized extracellular vesicles for cancer therapy: Potential theranostic applications in gastrointestinal tumors. Crit Rev Oncol Hematol 191:104121\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa Y, Dong S, Li X, Kim BYS, Yang Z, Jiang W (2020) Extracellular Vesicles: An Emerging Nanoplatform for Cancer Therapy. Front Oncol 10:606906\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMilette S, Sicklick JK, Lowy AM, Brodt P (2017) Molecular Pathways: Targeting the Microenvironment of Liver Metastases. Clin Cancer Res 23:6390\u0026ndash;6399\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatil S, Jir\u0026aacute;k D, Saudek F, H\u0026aacute;jek M, Scheffler K (2011) Positive contrast visualization of SPIO-labeled pancreatic islets using echo-dephased steady-state free precession. Eur Radiol 21:214\u0026ndash;220\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRenzulli M, Clemente A, Ierardi AM, Pettinari I, Tovoli F, Brocchi S, Peta G, Cappabianca S, Carrafiello G, Golfieri R (2020) 'Imaging of Colorectal Liver Metastases: New Developments and Pending Issues'. Cancers (Basel), 12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuiz-Cabello J, Barnett BP, Bottomley PA, Bulte JW (2011) Fluorine (19F) MRS and MRI in biomedicine. NMR Biomed 24:114\u0026ndash;129\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShao F, He R, Jin Y, Pang Y, Sun Z, Zhang B, Jin S (2025) 'Regulation and Clinical Application of Exosomes in Venous Thrombosis', \u003cem\u003eAngiology\u003c/em\u003e: 33197251319597\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSrivastava A, Rathore S, Munshi A, Ramesh R (2022) Organically derived exosomes as carriers of anticancer drugs and imaging agents for cancer treatment. Semin Cancer Biol 86:80\u0026ndash;100\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Y, Yang J, Li Y, Luo J, Sun J, Li D, Wang Y, Wang K, Yang L, Wu L, Sun X (2022) Single low-dose INC280-loaded theranostic nanoparticles achieve multirooted delivery for MET-targeted primary and liver metastatic NSCLC. Mol Cancer 21:212\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, Wei J, Nie G (2014) A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 35:2383\u0026ndash;2390\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWagner B, Drel V, Gorin Y (2016) Pathophysiology of gadolinium-associated systemic fibrosis. Am J Physiol Ren Physiol 311:F1\u0026ndash;f11\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWahsner J, Gale EM, Rodr\u0026iacute;guez-Rodr\u0026iacute;guez A, Caravan P (2019) Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem Rev 119:957\u0026ndash;1057\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWan L, Pantel K, Kang Y (2013) Tumor metastasis: moving new biological insights into the clinic. Nat Med 19:1450\u0026ndash;1464\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeng Q, Hu X, Zheng J, Xia F, Wang N, Liao H, Liu Y, Kim D, Liu J, Li F, He Q, Yang B, Chen C, Hyeon T, Ling D (2019) Toxicological Risk Assessments of Iron Oxide Nanocluster- and Gadolinium-Based T1MRI Contrast Agents in Renal Failure Rats. ACS Nano 13:6801\u0026ndash;6812\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu L, Wen X, Wang X, Wang C, Sun X, Wang K, Zhang H, Williams T, Stacy AJ, Chen J, Schmieder AH, Lanza GM, Shen B (2018) Local Intratracheal Delivery of Perfluorocarbon Nanoparticles to Lung Cancer Demonstrated with Magnetic Resonance Multimodal Imaging. Theranostics 8:563\u0026ndash;574\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu X, Zhang R, Liu F, Ping J, Wen X, Wang H, Wang K, Sun X, Zou H, Shen B, Wu L (2018) (19)F MRI in orthotopic cancer model via intratracheal administration of α(ν)β(3)-targeted perfluorocarbon nanoparticles. Nanomed (Lond) 13:2551\u0026ndash;2562\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Y, Hong Y, Cho E, Kim GB, Kim IS (2018) Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. J Extracell Vesicles 7:1440131\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng H, Guo S, Ren X, Wu Z, Liu S, Yao X (2023) 'Current Strategies for Exosome Cargo Loading and Targeting Delivery', \u003cem\u003eCells\u003c/em\u003e, 12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhan Q, Yi K, Qi H, Li S, Li X, Wang Q, Wang Y, Liu C, Qiu M, Yuan X, Zhao J, Hou X, Kang C (2020) 'Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy', \u003cem\u003eTheranostics\u003c/em\u003e, 10: 7889\u0026thinsp;\u0026ndash;\u0026thinsp;905\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang C, Yan K, Fu C, Peng H, Hawker CJ, Whittaker AK (2022) Biological Utility of Fluorinated Compounds: from Materials Design to Molecular Imaging, Therapeutics and Environmental Remediation. Chem Rev 122:167\u0026ndash;208\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang LY, Yang X, Wang SB, Chen H, Pan HY, Hu ZM (2020) Membrane Derived Vesicles as Biomimetic Carriers for Targeted Drug Delivery System. Curr Top Med Chem 20:2472\u0026ndash;2492\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Liver metastasis, ¹⁹F MRI, Exosome, Perfluorocarbon nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-7556946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7556946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e​​: Liver metastasis (LM) remains a leading cause of cancer mortality, with conventional imaging modalities suffering from physiological interference and insufficient sensitivity. Fluorine-19 (¹⁹F) MRI offers a promising solution due to its negligible endogenous background, yet existing targeted probes face limitations in tumor heterogeneity and receptor variability.\u003c/p\u003e\n\u003cp\u003e​​\u003cstrong\u003eMethods\u003c/strong\u003e​​: We developed exosome-biomimetic nanoparticles (EP-NPs) by coating perfluorocarbon nanoparticles (PFCE NPs) with exosomal membranes derived from HEK-293T cells. The hybrid platform combined the targeting capability of exosomes with the high sensitivity of ¹⁹F MRI. Characterization included dynamic light scattering, transmission electron microscopy, and ¹⁹F NMR. \u003cem\u003eIn vitro\u003c/em\u003e cellular uptake was evaluated in BT-549 and NCI-H446 cells using confocal microscopy. \u003cem\u003eIn vivo\u003c/em\u003e targeting efficiency and biodistribution were assessed in a BT-549 liver metastasis mouse model via ¹⁹F/¹H MRI. Toxicity was tested via MTT assays, serum biochemistry, and histopathology.\u003c/p\u003e\n\u003cp\u003e​​\u003cstrong\u003eResults\u003c/strong\u003e​​: EP-NPs exhibited a hydrodynamic diameter of 111.6 ± 8.2 nm, negative zeta potential (-20.50 mV), and stable ¹⁹F signal. Confocal imaging confirmed enhanced cellular uptake compared to non-targeted PFCE NPs. \u003cem\u003eIn vivo\u003c/em\u003e, EP-NPs enabled precisely and early detection of LM with sustained ¹⁹F signal. Biodistribution revealed accumulation in the liver and spleen, and toxicity assessments demonstrated no significant hepatorenal impairment or histological damage.\u003c/p\u003e\n\u003cp\u003e​​\u003cstrong\u003eConclusion\u003c/strong\u003e​​: EP-NPs integrate exosomal targeting and ¹⁹F MRI to achieve ultrasensitive LM detection with high biocompatibility and prolonged circulation. This platform holds potential for real-time monitoring of metastatic progression and clinical translation, overcoming limitations of conventional imaging agents.\u003c/p\u003e","manuscriptTitle":"Exosome-Biomimetic Nanoprobes Achieve Ultrasensitive Detection and Real-Time Monitoring of Liver Metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 05:07:33","doi":"10.21203/rs.3.rs-7556946/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T08:22:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-02T04:21:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T12:42:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201675986397494689142659193984705044757","date":"2025-09-26T02:51:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323530541590689337155740585979234045826","date":"2025-09-15T19:27:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T11:22:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-12T03:19:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T03:18:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Nanotechnology","date":"2025-09-07T14:29:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-nanotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cano","sideBox":"Learn more about [Cancer Nanotechnology](https://cancer-nano.biomedcentral.com/)","snPcode":"12645","submissionUrl":"https://submission.nature.com/new-submission/12645/3","title":"Cancer Nanotechnology","twitterHandle":"@CancerNanotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c4a040e-cb7a-4236-a4c4-6ecdb378391d","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:08:26+00:00","versionOfRecord":{"articleIdentity":"rs-7556946","link":"https://doi.org/10.1186/s12645-026-00367-0","journal":{"identity":"cancer-nanotechnology","isVorOnly":false,"title":"Cancer Nanotechnology"},"publishedOn":"2026-01-29 15:59:08","publishedOnDateReadable":"January 29th, 2026"},"versionCreatedAt":"2025-09-24 05:07:33","video":"","vorDoi":"10.1186/s12645-026-00367-0","vorDoiUrl":"https://doi.org/10.1186/s12645-026-00367-0","workflowStages":[]},"version":"v1","identity":"rs-7556946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7556946","identity":"rs-7556946","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}