Novel system for organ preservation in liver transplantation: Feed macrophages with ceria nanoparticles

Liver transplantation (LT) remains the primary treatment for irreversible liver failure. However, challenges in organ preservation, particularly ischemia-reperfusion injury and donor liver vulnerability, contribute to poor post-transplant outcomes.[1,2] The accumulation of reactive oxygen species (ROS) exacerbates these issues, leading to inflammation, tissue damage, and impaired graft function.[3] Ceria nanoparticles have gained attention for their antioxidant, antibacterial, and antifibrotic properties and exhibited multiple enzymatic activities, including those of superoxide dismutase and catalase.[4] We developed an innovative system for organ preservation using dextran-coated ceria nanoparticles (CeO2NPs) incorporated into the University of Wisconsin (UW) solution, aiming to enhance liver preservation by reducing oxidative stress and inflammation and adjusting macrophage polarization. Dextran-40k was selected to coat the ceria nanoparticles because it reduces hydrophobicity, improves dispersion, and prevents nanoparticle aggregation. This also helped to reduce the viscosity of the preservation solution, overcoming some limitations of the UW solution, such as its tendency to increase microcirculation resistance and promote erythrocyte aggregation. Analytical techniques such as Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy can confirm nanoparticle size, valence states, and catalytic activities, which are critical for their effectiveness in reducing ROS levels during organ preservation [Supplementary Figure 1, https://links.lww.com/CM9/C364]. After intravenous injection of 89Zr-DFO-CeO2NPs via the mice tail vein, the distribution of CeO2NPs in organs and cells was monitored at various intervals using positron emission tomography (PET) [Figure 1A]. The nanoparticles accumulated mainly in the liver, reaching peak concentrations between 8 h and 48 h post-injection, and only minimal presence beyond this period [Supplementary Figure 2A–C, https://links.lww.com/CM9/C364]. To identify the liver cells involved in CeO2NP uptake, CeO2NPs were labeled with fluorescein isothiocyanate (FITC) (green fluorescence) and injected into mice, and liver samples were collected at multiple time points. Merged immunofluorescence images showed FITC-labeled CeO2NPs localized within the macrophages, corroborating the PET imaging results [Figure 1B and Supplementary Figure 2A, https://links.lww.com/CM9/C364]. CeO2NPs uptake peaked between 8 h and 48 h post-injection, aligning with the highest nanoparticle concentration in the liver tissue. After 96 h, CeO2NPs were no longer detectable in the macrophages, indicating that they were metabolized between 48 h and 96 h after injection [Supplementary Figure 2D, https://links.lww.com/CM9/C364]. These results suggest that liver macrophages are primarily responsible for CeO2NP uptake and may play a critical role in their function.Figure 1: PET image at 20 min, 60 min, 8 h, 48 h, and 96 h after intravenous injection of 89Zr-DFO-CeO2NPs via the mice tail vein (A). Immunofluorescence staining data were also recorded at 20 min, 60 min, 8 h, 48 h, and 96 h from CeO2NP solution perfusion (B). Green marks the CeO2NP distribution, anti-CD68 (red) marks macrophages, and DAPI (blue) marks nuclear staining in liver tissues. Scale bars, 20 μm and 50 μm. H&E (C) and TUNEL (D) staining of liver samples consisted of 20× and 63× in all groups. IHC staining (20× and 63×) and incubation with primary rat anti-mouse F4/80 and Ly6G monoclonal antibodies (mAbs) demonstrated sterile inflammation and macrophage infiltration (E). IHC staining (20× and 63×) shows the potential macrophage polarization using primary rat anti-mouse iNOS and Arg1 mAbs (F). Immunofluorescence staining revealed anti-CD206 (green) antibody as the marker for M2, anti-iNOS (red) antibody as the marker for M1, and DAPI (blue) as the marker for nuclear staining in liver tissues (G). Scale bars, 20 μm and 50 μm. CeO2NPs: Dextran-coated ceria nanoparticles; DAPI: 4′,6-diamidino-2-phenylindole; H&E: Hematoxylin and eosin; IHC: Immunohistochemistry; iNOS: Inducible nitric oxide synthase; LPS: Lipopolysaccharide; LT: Liver transplantation; PET: Positron emission tomography; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.We designed 3 groups of mice that underwent different LT strategies (n = 5 in each group): the blank group (sham group), the ordinary LT group, and LT with the aid of CeO2NPs (CeO2NPs group). To explore its biological function in murine liver, mice underwent LT, then, liver function and tissue integrity were assessed via blood serum tests and staining assays. The serum levels of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase were measured [Supplementary Figure 3A, B, https://links.lww.com/CM9/C364]. The enzyme levels were significantly higher in the LT group than in the sham group, suggesting severe liver damage in untreated post-transplant mice. However, these levels were notably reduced in the CeO2NPs group, indicating a protective effect against liver injury. The LT group showed elevated levels of the oxidative stress markers, dihydroethidium (DHE), malondialdehyde (MDA) and reduced glutathione (GSH) levels [Supplementary Figure 3C, https://links.lww.com/CM9/C364]. By contrast, the CeO2NPs group showed no significant difference from the sham group in these markers, suggesting that CeO2NPs help mitigate oxidative stress in the liver after transplantation. Hematoxylin and eosin (H&E) staining revealed severe tissue damage and vacuolization in the LT group, while the CeO2NPs group showed much less damage, with histological characteristics similar to those of the sham group. A pathologist blinded to the groups evaluated the liver damage using Suzuki’s scoring system, confirming that CeO2NPs protected the liver from injury and maintained a stable tissue morphology [Figure 1C and Supplementary Figure 3D, https://links.lww.com/CM9/C364]. In addition to reducing liver enzyme levels and oxidative stress, CeO2NPs alleviated inflammation and cell death. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining revealed less necrosis and apoptosis in the CeO2NPs group than in the LT group, suggesting a protective effect on hepatocytes [Figure 1D and Supplementary Figure 3D, https://links.lww.com/CM9/C364]. To explore the role of macrophages in the inflammatory response, immunohistochemical staining was performed using an anti-F4/80 antibody [Figure 1E and Supplementary Figure 3D, https://links.lww.com/CM9/C364]. Macrophage infiltration was significantly less in the CeO2NPs group than in the LT group. The presence of fewer macrophages in the CeO2NPs group indicates that the nanoparticles helped control inflammation and mitigate immune responses following LT. Ly6G staining revealed higher neutrophil levels in the LT group, while the CeO2NPs group showed a significant reduction in neutrophil infiltration, further supporting the anti-inflammatory potential of CeO2NPs [Figure 1E and Supplementary Figure 3D, https://links.lww.com/CM9/C364]. Overall, these findings indicate that CeO2NPs can reduce liver injury, oxidative stress, and inflammation, thus providing a protective effect in murine LT. These nanoparticles show promise in mitigating ischemia-reperfusion injury and improving organ preservation outcomes. To investigate the role of sterile inflammation and macrophage polarization in murine liver samples, we analyzed cytokine profiles and macrophage phenotypes in relation to CeO2NPs distribution. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were elevated in the LT group, indicating a shift towards M1 macrophage polarization, whereas increased IL-10 levels were observed in the CeO2NPs-treated group, suggesting that the nanoparticles may promote an anti-inflammatory M2 phenotype [Supplementary Figure 4A, https://links.lww.com/CM9/C364]. Immunohistochemical staining using inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) revealed significantly higher iNOS expression in the LT group and higher Arg-1 expression in the CeO2NPs-treated group [Figure 1F and Supplementary Figure 4B, https://links.lww.com/CM9/C364], confirming an anti-inflammatory M2 phenotype and suggesting that CeO2NPs may be beneficial in reducing inflammation and supporting liver recovery by promoting M2 polarization. Previous serum analyses suggested that CeO2NPs improve liver function and reduce oxidative stress. We investigated the signaling pathways and mechanisms involved in this protective effect. Western blotting [Supplementary Figure 4C, https://links.lww.com/CM9/C364] revealed high expression of signal transducer and activator of transcription 1 (STAT1), which promotes M1 polarization, in the LT group. By contrast, phosphorylated STAT1 (p-STAT1), iNOS, TNF-α, and IL-1β were significantly reduced in the CeO2NPs-treated group, indicating a decrease in M1 polarization and a reduction in the proinflammatory environment. We also examined the role of the nuclear factor kappa B (NF-κB) pathway, a key regulator of both M1 and M2 macrophage phenotypes. Phosphorylation of p65, a subunit of NF-κB crucial for M1 polarization, was elevated in the LT group, suggesting a proinflammatory environment. By contrast, the CeO2NPs-treated group exhibited reduced p65 activity, which may help suppress M1 polarization and promote M2 polarization [Supplementary Figure 4D, https://links.lww.com/CM9/C364]. These results suggest that CeO2NPs modulate inflammation by balancing M1 and M2 polarization, thereby promoting an anti-inflammatory environment conducive to liver preservation. Our in vitro experiments supported these findings. Bone marrow-derived macrophages exposed to CeO2NPs phagocytosed the nanoparticles over time [Supplementary Figure 4E, https://links.lww.com/CM9/C364]. Immunofluorescence assays showed that, while lipopolysaccharide (LPS)-treated cells exhibited red fluorescence, indicative of M1 polarization, cells treated with both CeO2NPs and LPS showed predominantly green fluorescence, representing M2 polarization [Figure 1G]. Western blotting confirmed elevated iNOS and p-STAT1 levels in the LPS-treated group, indicating M1 polarization. However, p-STAT6 markedly increased in the CeO2NPs + LPS group, which was associated with M2 polarization [Supplementary Figure 4F, G, https://links.lww.com/CM9/C364]. These results suggest that CeO2NPs inhibit M1 polarization via the STAT1/NF-κB-p65 pathway and promote M2 polarization through the STAT6 pathway. In conclusion, CeO2NPs appear to reduce donor liver injury and sterile inflammation by inhibiting M1 polarization through the STAT1/NF-κB-p65 pathway and promoting M2 polarization via STAT6. This dual mechanism of action highlights the potential of CeO2NPs as a therapeutic agent to improve liver preservation and reduce post-transplant inflammation. This is a pioneering study documenting the use of CeO2NPs to improve liver preservation during transplantation. Previous research primarily focused on the antioxidant properties of CeO2NPs in normothermic perfusion models; while our study extended these findings to liver transplantation in mouse models.[5] Our results demonstrated that CeO2NPs not only reduced oxidative stress but also modulated cytokine release and macrophage polarization, leading to better graft function and reduced post-transplant complications. Further studies are warranted to optimize the use of CeO2NPs in clinical applications. Key areas of interest include investigating the dose-response relationship, exploring CeO2NP selectivity for hepatocytes, and understanding the interplay between different signaling pathways, such as STAT6 and NF-κB. Additionally, exploring the potential of CeO2NPs to mitigate fibrosis and improve long-term graft survival would provide valuable insights for future clinical applications. Acknowledgment We are grateful to Dr. Siddhartha Biswas (Greater Kailash Hospital, India) for his constructive criticism on the manuscript and revising English text. Conflicts of interest Patent number 202311078746X is still under consideration by the China National Intellectual Property Administration.