Specific therapeutic interventions of hepatic ischemia-reperfusion injury (IRI) to attenuate liver dysfunction or multiple organ failure following liver surgery and transplantation remain a key concern. Here we present an innovative strategy by integrating a platinum nanoantioxidant and nitric oxide synthase (iNOS) into the zeolitic imidazolate framework-8 (ZIF-8)-based hybrid nanoreactor for effective prevention of IRI. Platinum nanoantioxidant could scavenge excessive reactive oxygen species (ROS) at the injury site and meanwhile generate oxygen for subsequent synthesis of nitric oxide (NO) under the catalysis of iNOS. Such cascade reaction successfully achieved dual protection for the liver through ROS clearance and NO regulation, remarkably enabling reduction of oxidative stress, inhibition of macrophage activation and neutrophil recruitment, and ensuing suppression of proinflammatory cytokines. The current work establishes a proof of concept of multifunctional nanotherapeutics against IRI, which may provide a promising intervention solution in clinical use.
Article
Protective Effect of Platinum Nano-antioxidant and Nitric Oxide Against Hepatic Ischemia-Reperfusion Injury
https://doi.org/10.21203/rs.3.rs-660251/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 06 May, 2022
Version 1
posted 13 Jul, 2021
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hepatic ischemia-reperfusion injury
liver surgery and transplantation
multifunctional nanotherapeutics
Currently, liver resection and transplantation have been widely used in the clinic to treat various liver diseases such as intrahepatic bile duct stones, liver trauma, tumors and other diseases1. During the surgery, liver ischemia reperfusion injury (IRI) occurs due to the cessation and restoration of blood supply, which may result in an acute inflammatory response, severe liver damage, and even multiple organ failure and death2–4. Current intervention strategies for hepatic IRI include ischemic preconditioning, pharmacological agents preconditioning, gene therapy, and so on5,6. However, due to the complicated pathophysiological processes of IRI, there are still lack of effective solutions for the prevention and intervention of IRI in the clinic, so far.
Hepatic IRI is a pathological process involving multiple factors, including acidosis, oxidative stress, intracellular calcium overload, and activation of macrophages and neutrophils caused by hypoxic metabolism2,7. Pharmacological interventions such as supplementation of antioxidants to reduce the oxidative stress have be explored to minimize the risk of liver damage8–10. Recently, accumulating evidence reveals that nitric oxide (NO) plays diverse roles in modulating cell behaviors and NO-releasing materials have be designed for potential therapeutic applications11–15. In particular, it has been reported that the administration of inhaled NO, nitrite or NO donor drugs could attenuate ischemia/reperfusion injury during the liver surgery and accelerate the restoration of liver function16–18. However, challenges such as short half-life of NO, toxic side effect and drug tolerance remain and further improvements are needed14.
Since the discovery of Fe3O4 nanoparticles as the peroxidase mimics19, a variety of nanomaterials capable of mimicking the functions of natural enzymes, namely nanozymes, have attracted considerate interest20–22. Owing to advantages in stability, low cost, and recyclability, nanozymes have been widely used in areas of chemistry, biology, and medicine23–26. Among these nanozymes, several nanomaterials have displayed promising effect on the elimination of reactive oxygen species (ROS), which could be employed as biomimetic antioxidants to regulate ROS homeostasis25,27−30. In addition, nanozyme-catalyzed cascade reactions are becoming intriguing for versatile biomedical applications31–34. For example, Li et al integrated the artificial Au nanoparticle (NP) nanozyme and natural ATP synthase into hollow silica microspheres for mitochondria-mimicking oxidative phosphorylation31. In the designed natural-artificial hybrid architecture, the Au NPs could convert glucose into gluconic acid in the presence of oxygen (O2) and the resulting transmembrane proton gradient facilitated the production of ATP catalyzed by ATP synthase. Nanozyme-involved cascade reactions exhibit great benefits in reducing the diffusion barriers, minimizing intermediate decomposition and enhancing local concentrations of reactants, thereby improving the intercommunication and efficiency of catalytic reactions33,34.
Given the endogenous synthesis of NO produced from ʟ-Arginine (ʟ-Arg) by the catalysis of nitric oxide synthases (NOS), herein, we rationally designed a nanozyme-containing biomimetic cascade system to achieve simultaneous NO generation and noxious ROS depletion for effective prevention of IRI. To make full use of the inherent advantages of nanozymes and natural enzymes, ultrasmall platinum nanoparticles (Pt NPs) with superoxide dismutase/catalase-like properties and induced nitric oxide synthase (iNOS) are integrated into the zeolitic imidazolate framework-8 (ZIF-8) carriers to form a safe and effective nanoreactor ([email protected]). By virtue of ROS produced in the process of hepatic IRI as the reactant, the Pt NPs nanozyme could not only achieve effective ROS elimination, but also result in a huge amount of oxygen (O2) generation, which further promotes the production of NO via the catalysis of iNOS in the presence of ʟ-Arg (Fig. 1a). With the aid of [email protected], the whole process can greatly reduce oxidative stress-induced damage, inhibit cell apoptosis and reduce the expression of proinflammatory cytokines, leading to effective intervention of hepatic IRI (Fig. 1b). Overall, the designed nanoractor not only improves the targeting and bioavailability of NO, but also exhibits dual protective effects via ROS elimination and NO modulation, thereby offering a promising strategy to prevent the liver from IRI.
Design, synthesis, and characterization of [email protected] In this study, the natural-artificial hybrid nanoreactor was designed and synthesized. First, ultrasmall polyvinylpyrrolidone-coated Pt NPs were successfully synthesized according to the reported method35. Then the co-precipitation approach was applied to simultaneously embed enzyme molecules (iNOS) and Pt NPs into the ZIF-8 supporting matrix (Fig. 2a). Transmission electron microscopy (TEM) and elemental mapping results clearly indicated that the obtained Pt NPs had an average size of ~ 3 nm with a narrow size distribution. Interestingly, the Pt NPs could be effectively deposited in the whole ZIF-8 NPs during the co-precipitation process (Fig. 2b-d). The as-prepared [email protected] displayed an average size distribution of 99.7 ± 9.0 nm by dynamic light scattering (DLS) measurement (Fig. 2e). Next, TEM results suggested the loading content of Pt element gradually increased with the increasing feeding amount of Pt NPs (Pt:Zn ratios of 1:250, 1:50, and 1:10) (Supplementary Fig. S1). The optimal loading content of Pt in [email protected] with well dispersibility in aqueous solutions was calculated to be 2.3% (wt %) by inductively coupled plasma-optical emission spectrometry (ICP-OES). To investigate the enzyme-mediated biomimetic mineralization processes, the [email protected] NPs were synthesized, which displayed similar morphology with ZIF-8 NPs. X-ray diffraction (XRD) pattern suggested no significant difference in the crystal structure before and after the enzyme encapsulation (Fig. 2f and Supplementary Fig. S2). In addition, Fig. 2g showed that there were close zeta potential values before and after the iNOS encapsulation, further validating the enzyme embedding, rather than surface absorption processes36. The zeta potential of [email protected] was about 8.6 mV and its favored dispersibility in aqueous solutions with maximum loading content of iNOS up to 6.8 wt% is promising for further in vivo applications.
In vitro performance. Firstly, the enzyme-like catalytic activities of Pt NPs were examined by superoxide dismutase (SOD) activity assay kit and hydrogen peroxide assay kit, respectively. SOD is known to catalyze the superoxide anion (•O2− ) to generate oxygen (O2) and hydrogen peroxide (H2O2), while catalase (CAT) is able to decompose H2O2 into H2O and O2. By following the commercially available standard protocols, the inhibition rate of •O2− and H2O2 was obtained. Results demonstrated the elimination ability of both •O2− and H2O2 increased with the increase of Pt NPs concentrations (Fig. 3a, 3b). To further verify the SOD/CAT mimic capability, the time course of O2 generation was monitored by dissolved oxygen probe. In the presence of Pt NPs, the O2 level gradually increased over time and the addition of more Pt NPs would undoubtedly accelerate the generation of O2 (Fig. 3c). Moreover, the Michaelis-Menten constant (KM) and maximum velocity (Vmax) were determined to be 172 mM and 3.46 × 10− 6 M s− 1, given the Michaelis-Menten curves and Lineweaver-Burk plot (Fig. 3d, 3e). Taken together, these results clearly demonstrated that Pt NPs exerted both superior SOD and CAT-like activities, enabling effective elimination of •O2− and H2O2.
Given the consumption of O2 during the synthesis of NO, we subsequently evaluated whether the generated O2 could continuously promote the oxidization of ʟ-Arg into NO. Compared to [email protected] and [email protected], the integration of both Pt NPs and iNOS enzyme exhibited better catalytic activities of NO production. Additionally, a positive correlation between NO level and the loading amount of Pt NPs was obtained (Fig. 3f). To evaluate the overall antioxidant capacity of the as-prepared [email protected] nanoreactor, the 2,2’-Azino-bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) assay was performed based on the reduction of ABTS•+ radicals by antioxidants (Fig. 3g). As shown in Fig. 3h, the ABTS•+ radicals with blue color exhibited obvious absorbance decrease and discoloration in the presence of [email protected] Also, the radical scavenging efficiency is positively related to the nanoparticle concentrations, implying favorable antioxidant ability of [email protected] to scavenge ATBS radicals (Fig. 3i).
Cellular evaluation of [email protected] Next, we examined the antioxidant effects of [email protected] in primary mouse hepatocyte FL38B cells. H2O2 was utilized to stimulate intracellular oxidative stress and 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was applied as ROS indicator. As shown in Fig. 4a, strong green fluorescence signals were observed with the addition of H2O2 as compared to the control group without H2O2 treatment. When treated with [email protected] or [email protected], the intracellular fluorescence signals were remarkably decreased, while there was minimal fluorescence disturbance in the ZIF-8 NPs treated group. Quantitative analysis by flow cytometry revealed reduced intracellular ROS level (51.4%) in ([email protected] + H2O2)-treated cells as compared to that in H2O2-stimulated cells (59.1%), suggesting the ROS scavenging effect from Pt nanozyme. As comparison, [email protected] treated group remarkably decreased ROS level to around 22.8% in H2O2-pretreated cells (Supplementary Fig. S3). Moreover, the [email protected] exhibited concentration-dependent inhibition of ROS generation (Fig. 4c). Subsequently, intracellular NO levels were evaluated by the nitric oxide indicator (DAF-FM diacetate) via flow cytometry analysis and [email protected] + H2O2 group exhibited stronger fluorescence signals than the other groups (Fig. 4b). Then the protective effect of [email protected] against H2O2-induced oxidative stress in cells was evaluated by the standard methyl thiazolyl tetrazolium (MTT) assay. Unsurprisingly, H2O2 (250 µM) treatment resulted in about 41% cell death after incubation for 24 h, while the addition of [email protected] exhibited concentration-dependent increase of the cell viability, implying its protective effect against oxidative damage (Fig. 4d). Moreover, all designed NPs would induce negligible cell death in hepatocyte, HEK 293 cells and macrophage cells (up to 40 µg/mL), suggesting their good biocompatibility and minimum side effect from the nanoformulas themselves (Supplementary Fig. S4-S8). Collectively, all these results demonstrated that [email protected] NPs could effectively alleviate oxidative stress and rescue cells from ROS-induced damage.
In vivo imaging and biodistribution. In comparison with small molecular drugs, various types of nanomaterials have intrinsic advantages of passive targeting and accumulation in the liver. To evaluate the biodistribution and liver accumulation of [email protected] in living systems, Cy5 labeled iNOS enzyme was applied to form Pt-iNOS (Cy5)@ZIF and real-time fluorescence imaging was carried out in mice after intravenous injection (i.v.) of the NPs. As shown in Fig. 5a and Supplementary Fig. S9, there was an obvious increase of fluorescence signals at 1 h post injection (p.i.) and semi-quantitative analysis revealed that the NPs could remain in the liver for up to 10 h (7.2 × 108 p/s/cm2/sr) and were gradually cleared from the liver by 24 h (3.4 × 108 p/s/cm2/sr) p.i. Moreover, the ex vivo biodistribution in major organs at 24 h verified by fluorescence imaging and ICP-OES demonstrated that the NPs were able to efficiently accumulate in the liver (22.84 ± 3.65%ID g− 1), serving as an optimal candidate for IRI intervention (Fig. 5b-d).
Protective effect in a hepatic IRI model. Based on the promising antioxidant effect of [email protected], we further investigated the feasibility of the protective effect against IRI in a murine model. C57BL/6 mice were treated with different nanoplatforms for 12 h before the clamping of porta hepatis. After 60 min of ischemia and reperfusion, the blood and liver samples with various treatments were collected and liver functions were further evaluated at 12 h after surgery. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were clinically applied as indicators of liver damage. Compared to the sham-operated control group, the IRI mice group displayed significant increase of ALT and AST levels, indicating alleviated liver injury. When the IRI mice were pretreated with different types of NPs, the [email protected] treated IRI mouse group had remarkably reduced ALT and AST levels compared to those in ZIF-8 NPs, Pt NPs and [email protected] treated groups, clearly demonstrating the effective intervention of liver injury by [email protected] (Fig. 5e and 5f). To further evaluate the therapeutic efficacy, hematoxylin and eosin (H&E) staining was performed in liver tissues after pretreatment with various NPs in IRI mice. As expected, severe hepatocyte necrosis and cytolysis was observed in the PBS-treated IRI group after 12 h. The ZIF-8 NPs, Pt NPs and [email protected] NPs treated groups displayed indistinguishable difference in the liver injury. By contrast, [email protected] IRI group remarkably reduced the hepatocyte damage, implying the effective prevention from IRI (Fig. 6a). Overall, the results were consistent with the trend of ALT and AST levels. Moreover, the difference between [email protected] treated IRI group after 7 days and the control group was marginal (Supplementary Fig. S10). Besides the therapeutic effectiveness of the designed nanomedicine, the safety in living systems is also important for further potential clinic applications. Hemolysis test (24 h p.i.), profiles of liver and kidney functions, and H&E staining of major organs after injection of [email protected] for 7 days were evaluated (Supplementary Fig. S11-S14). All results showed [email protected] exhibited excellent biocompatibility and could be considered as an ideal candidate for the prevention of IRI.
Liver IRI is a pathophysiological event and oxidative stress is considered as one essential mechanism. To further obtain visual evidence for cellular events, immunofluorescence imaging was performed on liver tissues. Results indicated that hepatic IRI led to obvious activation of monocytes/macrophages compared to the sham group. The designed [email protected] NPs can slightly inhibit the activation of macrophages, likely due to its ROS scavenging effect (Fig. 6b). By employing both ROS scavenging derived from Pt nanozyme and NO-based modulating effect, [email protected] treated mice exhibited minimal activation of monocyte/macrophages. Similarly, the [email protected] treated group also greatly suppressed the recruitment of neutrophils and caspase activities for preventing cells from apoptosis compared to the IRI group (Fig. 6c and Supplementary Fig. S15). Taken together, all these findings indicated that the preconditioning of [email protected] could inhibits IRI-induced macrophage activation, neutrophil accumulation, and subsequent apoptotic processes.
Encouraged by the above results, we further investigated the anti-inflammatory activities of the designed [email protected], the expression of mRNAs for the pro-inflammatory mediators, including interleukin-1 beta (IL-1β), interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon gamma (INF-γ) were measured from each group. As shown in Fig. 7, the levels of these pro-inflammatory cytokines were significantly increased in the hepatic IRI group, while they were reduced to relatively normal ranges in [email protected] treated IRI group. TNF-α is one intensively studied cytokine in response to inflammatory and immunomodulatory stimuli, and is a regulator responsible for the production of ROS. Also, IL-1 can facilitate the synthesis of TNF-α by Kupffer cells and recruitment of neutrophils 37. IFN-γ produced mainly by activated natural killer T cells will promotes Kupffer cells or dendritic cell activation. The Kupffer cells and neutrophil activation in turn will induce the release of various chemokines and cytokines, including TNF-α, IL-1β, IL-6, IL-12, etc.38, which further activates local immune cells, recruits circulating immune cells and aggravates liver damage. Taken together, these results indicated that [email protected] preconditioning would significantly suppress the expression of proinflammatory cytokines, which is generally involved in the initiation and propagation of IRI.
IRI include a series of complicated processes including the restriction of blood supply, subsequent restoration and reoxygenation, during which the imbalance of metabolic supply, inflammation and oxidative damage are involved. Additionally, the innate and adaptive immune responses will be activated, resulting in cell damage and organ dysfunction2. In-depth understanding of the molecular mechanisms of IRI and innovative intervention strategies will greatly improve the success of surgery and survival rate of patients. Oxidative stress is considered to play a pivotal role in ischemia and reperfusion damage. It has been reported that ROS scavenging via supplementation of antioxidants has the ability of diminishing oxidative stress and ensuing organ injury39. Antioxidant enzyme-SOD can promote the conversion of •O2− to O2 and H2O2. Also, H2O2 can be decomposed to H2O and O2 under the catalysis of CAT. Therefore, both enzymes exhibited beneficial actions by accelerating the detoxification of ROS in IRI. However, the short half-life of these natural proteases in the body (half-life of SOD is about 6 min), difficulty of cell uptake, and low delivery efficiency hinder their further applications8. Besides, NO, one of therapeutic gaseous molecules, has multiple regulatory functions, such as improving the microcirculation, suppressing caspase activities, and inhibiting neutrophil infiltration and platelet aggregation40. NO-based therapy has been applied for pulmonary hypertension and cardiopulmonary disorders for many years41, and its therapeutic effect on protection liver from IRI has increasingly received considerable attention17,42. Although many approaches have been explored in the prevention of IRI, there are still no ideal intervention strategies applied in clinical practice.
More recently, Pt nanozyme has attracted great interest as the substitute of natural enzymes43,44. In this study, we successfully synthesized ultrasmall Pt NPs, which could act as SOD and CAT mimics with excellent ROS scavenging capacity. In order to further enhance the therapeutic efficacy, NO-based therapy is incorporated by integrating the iNOS enzyme and Pt nanozyme into ZIF-8 architecture to achieve synergistic effect. Co-precipitation method was applied to obtain the hybrid nanoreactor, ensuring efficient embedding of iNOS into ZIF-8 during the process of nanoparticle formation. Such design not only prevents natural enzymes from inactivation and degradation in the body, but also promotes the molecular diffusion and intercommunication due to the intrinsic porous feature and large surface areas of metal-organic frameworks45,46. More importantly, as the synthesis of NO is O2-dependent, the generated O2 by Pt catalysis will further improve the iNOS-mediated cascade reaction activities. After the ischemia and reperfusion, the restoration of blood flow will cause the release of ROS, activation of macrophage cells and recruitment of neutrophils, which further leads to the release of large amount of chemokines and cytokines2. Apart from the ROS elimination by Pt nanozyme, protective actions of NO during ischemia and reperfusion are probably attributed to antioxidant and anti-inflammatory effects. The prepared [email protected] nanoreactor could maintain ALT and AST levels of IRI mice in the relatively normal range. Additionally, it greatly suppressed the activation of macrophages cells, along with the minimized infiltration of neutrophils and expression of pro-inflammatory cytokines, which eventually attenuated the liver damage caused by IRI.
In summary, we developed an ZIF-8-based hybrid nanoreactor, which encapsulated the Pt nanozyme and natural enzyme iNOS, for protecting the liver from IRI. By using the excessive ROS generated during hepatic ischemia/reperfusion as a stimulus, the Pt nanozyme with SOD/CAT-like properties could scavenge •O2− and H2O2 to produce O2, which can further react with ʟ-Arg to form NO under the catalysis of iNOS enzyme. Such designed cascade reaction successfully achieved the ROS scavenging (•O2− and H2O2) and NO production, which effectively reduced oxidative stress and expression of proinflammatory cytokines in the process of hepatic ischemia and reperfusion. Overall, this study may shed new light on dual protection mechanism of ROS clearance and NO regulation, which is beneficial to advance further clinical applications in hepatic IRI.
Syntheis of platnium nanoparticles (Pt NPs). The ultra-small platinum nanoparticles were prepared according to the reported method with slight modification35: Briefly, in 10 mL of 1 mM hexachloroplatinic acid hexahydrate (H2PtCl6•6H2O) solution, 111.1 mg of polyvinylpyrrolidone (PVP) was added. After stirring for 15 min, 200 µL freshly prepared sodium borohydride (NaBH4) solution (100 mM) was added and stirred for 12 h. The solution turned dark brown and Pt NPs were obtained for further use.
Syntheis of ZIF-8, [email protected], [email protected] Nanosystms. The ZIF-8 nanoparticles were synthesized according to the previous method36. Briefly,100 µL Zn(NO3)2•6H2O aqueous solution (0.5 M) was added into 900 µL 2-methylimidazole (2-MIN, 3.5 M) and the mixture was stirred at room temperature for 1 h. The obtained product was collected by centrifugation (8000 rpm, 10 min) and washed with water for three times. Similarly, nanoscale [email protected] was synthesized by mixing the 50 µL Pt NP solution with 900 µL 2-methylimidazole, followed by the additon of Zn(NO3)2•6H2O, The resulting [email protected] was redispersed in water for further use. To synthezie nanoscale [email protected], 0.3 mg iNOS enzyme was incubated with 900 µL 2-MIN for 10 min at 30°C followed by addition of zinc nitrate. For the synthesis of Pt-iNOS(Cy5)@ZIF, 0.5 mg cyanine 5 NHS ester (Lumiprobe) was reacted with iNOS (0.3 mg) in PBS solution for overnight. Then the product was dialyzed to remove excessive dye molecules. The obtained iNOS (Cy5) was used to prepare Pt-iNOS(Cy5)@ZIF by using the same method for the synthesis of [email protected] After centrifugation and washing with water for three times, the obtained products were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS).
Assays of SOD-like and CAT-like activities. To evaluate the Pt nanozyme properties, the superoxide anion scavenging activity was conducted with a SOD assay kit (Sigma-Aldrich, USA). The hydrogen peroxide quenching activity was performed with the Amplex® red hydrogen peroxide assay kit (Sigma-Aldrich, USA). All expreiments were carried out according to the standard protocol. To monitor the O2 production, different concentrations of Pt NPs (0.5, 1.0 and 2.0 µg/mL ) were mixed with 10 mM H2O2 in PBS buffer (pH 7.4), the dissiolved oxygen levels over time were measured by a dissolved oxygen probe. To obtain the enzyme kinetic parameters, different concencentraions of H2O2 were incubated with Pt NPs (2.0 µg/mL) for 10 min, the Michaelis-Menten constant (KM) and maximum velocity (Vmax) were determined as following equation:
In vitro measurement of total antioxidant capacity. The ABTS•+ radical scavenging assay was applied to evaluate the total antioxidant capacity of [email protected] Briefly, 7 mM ABTS in deionized water was prepared and reacted with 2.45 mM potassium persulfate overnight. Then the solution turned dark blue and ABTS radical cation (ABTS•+) was obtained. Next, 200 µL diluted solution was mixed with different concentrations of [email protected] for 5 min and the UV-Vis spectra were recorded. The radical scavenging efficiency was calculated based on the absorbance changes at 734 nm.
Intracellular ROS and NO measurements. Intracellular ROS levels were measured using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) as the ROS indicator and NO levles were measured using nitric oxide indicators (DAF FM Diacetate). 8 µg/mL different types of NPs (ZIF-8, [email protected], [email protected]) were added into cells and cultured for 2 h, followed by the treatment with H2O2 (1 mM) and incubated for another 4 h at 37°C. Then, the cells were stained with DCFH-DA (10 µM) or DAF probe (5 µM) for 30 min. After removing the excessive probe, fluoresence images were acquired by confocal laser scanning microscopy (CLSM). H33342 channel (λex = 408 nm ), FITC channel (λex = 488 nm). Quantative ROS levels with dfferent concentrations of [email protected] treatment (1, 2, 4, 8 µg/mL) were analyzed by flow cytometry.
Preparation of hepatic IRI model in mice. Male C57BL/6 mice (6–8 weeks old) were received from Zhejiang Experimental Animal Center and were fed with a standard diet and water. All animal laboratory operations were carried out according to the Guide of Animal Ethics Committee of Shanghai Skin Disease Hospital. For hepatic IRI model preparation, the mice were fasted for 12 h before the surgical operation. After the mice were anesthetized with isoflurane, they were placed on a heated surgical pad and the abdomen of the depilated mice was disinfected with iodophor solution. Then a midline laparotomy was performed to expose the portal triplet. The portal triplet was carefully lifted using a vessel forceps, and all structures in the portal triplet (hepatic artery, portal vein, and bile duct) were blocked using a microvascular clamp. The abdominal wall was covered with PBS-soaked gauze and the blocking process lasted 60 min. After 60 min, microvascular clamp was removed for reperfusion. Signs of the reperfusion can be observed by the immediate color change of the central lobe and the left lobe of the liver.
In vivo imaging and biodistribution. For the fluorescence imaging, 100 µL Cy5-labled nanocomposite (Pt-iNOS(Cy5)@ZIF, 2 mg/kg) solutions was intravenously injected into male C57BL/6 mice (6–8 weeks old). In vivo fluorescence imaging was recorded at 1, 4, 10 and 24 h p.i. on an IVIS Spectrum system. At 24 h post injection, the above mice were sacrificed, and major organs were collected and washed before optical imaging. The fluorescence intensity of livers was acquired from the analysis of the region of interest (ROI) using a Living Image software.
Protective effect in a hepatic IRI model. The mice were randomly divided into six groups (n = 5) different formulations: (1) Sham, (2) PBS + IRI, (3) ZIF-8 + IRI, (4) Pt NPs + IRI, (5) [email protected] + IRI, (6) [email protected] + IRI. Nanoformulations (2 mg/kg) in each group were intravenously injected 12 h before surgical operation. After 12 h induction of the hepatic IRI model in mice, the blood was collected for biochemical analysis. Liver and kidney functions were evaluated by blood tests of alanine aminotransferase (ALT) levels and aspartate aminotransferase (AST) levels (Shanghai Institute of Materia Medica, Center of Drug Safety Evaluation Research, CDSER, SIMM).The left liver lobes were dissected for hematoxylin and eosin (H&E) and immunofluorescence staining.
Immunofluorescence staining. Frozen tissue sections were prepared and covered with OCT media. Then the liver tissue sections were fixed with zinc fixative solutions for 10 min. After rinsing slides with PBS for 3 times, the liver tissues were further treated with 2% Triton X-100 for 15 min. Then 100 µL blocking buffer (10% FBS in PBS) was added onto tissues for 1 h. Antibodies including F4/80, CD31, Ly6G and caspase-3 were diluted and added on slides and incubated for overnight at 4°C. To stain the secondary antibody, slides were washed and incubated with secondary antibody conjugated with Alex 488 or 594 for 1 h. After that, the slides were rinsed with PBS and mounted with DAPI-containing mounting solution. Fluorescence images were acquired via confocal microscopy.
Measurement of inflammatory cytokine levels in liver tissues. To evaluate inflammatory cytokine levels,the left liver lobe tissues in each group were harvested at 12 h after surgery to detect the relative mRNA expression of IL-1α, IL-1β, TNF-α, IFN-γ, IL-12 and IL-6 using quantitative real-time polymerase chain reaction (qPCR) assay. Total RNA was extracted from the obtained liver tissues and reverse transcripted into cDNA in a gradient RNA apparatus. Subsequently, fluorescence qPCR amplification was performed and the relative mRNA expression levels of the cytokines mentioned above were measured and calculated.
Statistical analysis
Quantitative data were presented as mean ± s.d. Statistical differences were calculated by an unpaired two-tailed Student’s t-test using Excel and A One-way analysis of the variance (ANOVA) using GraphPad Prism software. P values < 0.05 were considered statistically significant and illustrated by *P < 0.05, ** P < 0.01, *** P < 0.001, respectively.
Data availability
The authors declare that the data supporting the findings of this study are available within the Article and its Supplementary Information Files or from the corresponding author upon reasonable request.
Acknowledgements
This work was financially supported by the Basic Research Program of Shenzhen (JCYJ20180305163622079), National University of Singapore Start-up Grant (NUHSRO/2020/133/Startup/08) and NUS School of Medicine Nanomedicine Translational Research Programme (NUHSRO/2021/034/TRP/09/Nanomedicine). All animal experiments were conducted according to the Guide of Animal Ethics Committee of Shanghai Skin Disease Hospital and the Animal Ethical and Welfare Committee of Shenzhen University (AEWC-SZU).
Author contributions
J. M., L. H., P. H., Y. Y., and X. C. conceived and designed the project. J. M., L. H., G. L., and J. Z. performed the material synthesis and characterizations. D. Z. and C. J. performed the XRD and elemental mapping studies. J. M., Y. S. performed the cell studies. J. M., C. L., and L. H. performed the animal studies. J. M., C. L., X. W., P. H., and Y. Y. analyzed animal results. J. M., C. L., L. H., P. H., Y. Y., and X. C. analyzed the results and co-wrote the paper. All the authors have discussed the results and approved the final version.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary Information for this paper is available at www.nature.com/xxx.
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There is NO Competing Interest.
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Reporting Summary