AuCePt porous hollow cascade nanozymes targeted delivery of disulfiram for alleviating hepatic insulin resistance

AuCePt porous hollow cascade nanozymes targeted delivery of disulfiram for alleviating hepatic insulin resistance | 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 AuCePt porous hollow cascade nanozymes targeted delivery of disulfiram for alleviating hepatic insulin resistance Huawei Shen, Yafei Fu, Feifei Liu, Wanliang Zhang, Yin Yuan, Gangyi Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4580829/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Oct, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract As the pathophysiological basis of type 2 diabetes mellitus (T2DM), insulin resistance (IR) is closely related to oxidative stress (OS) and inflammation, while nanozymes have a good therapeutic effect on inflammation and OS by scavenging reactive oxygen species (ROS). Hence, AuCePt porous hollow cascade nanozymes (AuCePt PHNs) are designed by integrating the dominant enzymatic activities of three metallic materials, which exhibit superior superoxide dismutase/catalase-like activities, and high drug loading capacity. In vitro experiments proved that AuCePt PHNs can ultra-efficiently scavenge endogenous and exogenous ROS. Moreover, AuCePt PHNs modified with lactobionic acid (LA) and loaded with disulfiram (DSF), named as AuCePt PHNs-LA@DSF, can significantly improve glucose uptake and glycogen synthesis in IR hepatocytes by regulating the insulin signaling pathways (IRS-1/AKT) and gluconeogenesis signaling pathways (FOXO-1/PEPCK). Intravenous administration of AuCePt PHNs-LA@DSF not only showed high liver targeting efficiency, but also reduced body weight and blood glucose and improved IR and lipid accumulation in high-fat diet-induced obese mice and diabetic ob/ob mice. This research elucidates the intrinsic activity of AuCePt PHNs for cascade scavenging of ROS, and reveals the potential effect of AuCePt PHNs-LA@DSF in T2DM treatment. Insulin resistance Oxidative stress Nanozymes Disulfiram Targeted drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Metabolic diseases such as obesity, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) are the most common metabolic diseases at present, with billions of patients worldwide, and the incidence rate is increasing rapidly. In China, the prevalence of NAFLD has exceeded 30%, and the prevalence of T2DM has exceeded 11.5%, which has become a major public health problem and a serious social medical burden [ 1 – 3 ]. The pathophysiological basis of the occurrence and development of these metabolic diseases is metabolic disorder and insulin resistance (IR) [ 4 ]. However, to combat metabolic diseases, it is necessary to use safe and effective drugs for a long time. Ideal drugs can not only improve metabolic disorders in vivo but also ameliorate insulin sensitivity and have better targeting and fewer side effects on metabolic tissues and organs. In the past 50 years, although extensive research has been carried out and some progress has been made, there is still no ideal drug for clinical application in the treatment of metabolic diseases. Therefore, it is necessary to adopt new approaches and drugs to restore systemic metabolic homeostasis to help cope with the increasingly serious public health crisis caused by metabolic diseases. Oxidative stress (OS) is one of the important reasons for the occurrence and development of IR [ 5 – 7 ], which is caused by the imbalance between the production and clearance of reactive oxygen species (ROS) and reactive nitrogen species (RNs). Studies have shown that OS can activate and induce IκB kinase (IKK)/NF-кB and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathways, interfere with the signal transduction of insulin receptors (InsR) and the phosphorylation of downstream signaling molecules such as insulin receptor substrate 1 (IRS-1) and protein kinase B (AKT), and inhibit the expression of glucose transporters, leading to glucose metabolism disorder and IR [ 8 – 10 ]. Therefore, one of the effective ways of improving IR to ameliorate antioxidant activity is by eliminating excessive ROS and reducing OS in vivo. With the development of nanotechnology, various nanomaterials have been developed and widely used in the diagnosis and treatment of diseases [ 11 , 12 ]. Targeted delivery of drugs has become a hotspot in tumor research, which increases the concentration and efficiency of drugs in the lesion site and reduces the toxic and side effects caused by nonspecific distribution of drugs [ 13 , 14 ]. Nanozymes, another research hotspot in nanoscience, are an umbrella term for a class of nanomaterials with similar biological enzymatic activities [ 15 , 16 ]. They can simulate the activity of one or more biological enzymes, such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT). For instance, Au nanomaterial can simulate the activities of POD, CAT, and SOD. More importantly, Au nanomaterial boasts high stability and biocompatibility and can be functionally modified via gold-ammonia bonds and gold-sulfur bond [ 17 ]. The H 2 O 2 adsorption affinity of the Pt (111) plane is higher than that of the Au (111) plane, which endows the Pt nanomaterial with higher catalytic activity [ 18 ]. Ce nanomaterials also have excellent SOD-, CAT-, and oxidase-like activities owing to the reversible conversion of their ionic state between Ce 3+ and Ce 4+ [ 19 ]. However, the catalytic activity and biological function of single-component nanomaterials are relatively limited [ 20 ]. Thus, if the advantages of the three (Au/Ce/Pt) components can be combined, the catalytic activity and biological functions of nanozymes can be notably enhanced. For many years, disulfiram (DSF), which was originally found to be an inhibitor of nuclear factor-κB (NFκB), has been widely used to treat alcoholism. It has anti-inflammatory properties and can treat a variety of cancers [ 21 , 22 ]. A recent study found that DSF treatment resulted in weight loss and improved insulin sensitivity and liver lipid accumulation in mice fed a high-fat diet (HFD) [ 23 ]. Although there is a preliminary understanding of the role of DSF in metabolic regulation, previous studies have been relatively shallow observational studies. In particular, DSF lacks targeting to major metabolic organs, such as the liver. Therefore, the development of DSF vectors targeting the liver has important clinical application value for the treatment of metabolic disorders. In the current study, we constructed a novel nanozyme capable of cascading ROS scavenging, namely AuCePt porous hollow cascade nanozymes (AuCePt PHNs), which can simulate SOD/CAT-like activities to catalyze a cascade of enzyme reactions, thereby efficiently scavenging ROS and inhibiting OS. AuCePt PHNs were modified by lactobionic acid (LA) and loaded with DSF (AuCePt PHNs-LA@DSF) for targeted hepatocyte delivery and combined treatment of hepatic IR (Scheme 1 ). We found that AuCePt PHNs-LA@DSF could improve glucose uptake and glycogen synthesis of IR hepatocytes by activating the IRS-1/AKT signal pathway and inhibiting the forkhead box O1 (FOXO-1)/phosphoenolpyruvate carboxykinase (PEPCK) signal pathway. It was also found that the intravenous injection of AuCePt PHNs-LA@DSF reduced body weight and blood glucose levels and improved hepatic IR in HFD-fed mice and leptin-deficient (ob/ob) diabetic mice. In general, our research results revealed the beneficial role of AuCePt PHNs-LA@DSF in controlling blood glucose, improving hepatic insulin sensitivity and lipid deposition, and suggested its potential efficacy in the treatment of OS-related metabolic diseases. Experimental Section Synthesis of AuCePt PHNs AuCePt PHNs were synthesized according to previously reported methods with some modifications [ 24 ]. Briefly, Na 3 C 6 H 5 O 7 solution (400 µL, 0.1 M) and NaBH 4 (400 µL, 1.0 M) were added to the preprepared dissolved oxygen-depleted ddH 2 O (100 mL) and stirred for 5 min. With the addition of cobalt chloride solution (100 µL, 0.5 M), the solution color rapidly changed from colorless to gray. Immediately, HAuCl 4 solution (200 µL, 0.1 M) was added dropwise. After stirring continued for 20 min, the nitrogen gas was stopped. When the color of the solution changed from gray‒red to dark blue, Ce(NO 3 ) 3 (100 µL, 0.1 M) and K 2 PtCl 6 (100 µL, 0.1 M) were added into the mixture and continuously stirred for 30 min. Finally, AuCePt PHNs were collected by centrifugation and washed three times with deionized water. Preparation of AuCePt PHNs-LA Because L-cysteine (L-Cys) has both sulfhydryl and amino groups, it was used as an intermediate molecule to connect AuCePt PHNs and LA through gold-sulfur bonds and amide bonds [ 25 ]. As-prepared AuCePt PHNs (40 mg) were dispersed into a freshly prepared L-cys solution (10 mL, 1 mM). After vigorous stirring for 3 min, the mixture was allowed to stand at 4°C for 16 h. Next, the mixture was washed with deionized water three times to remove the excess L-cys ligands. LA (8 mg), EDC⋅HCl (4.7 mg) and NHS (2.8 mg) were dissolved in 1 mL deionized water and stirred for 3 h. The above solution was mixed with AuCePt PHNs-L-cys (9 mL) and then stirred for 16 h at 4°C to construct AuCePt PHNs-LA. Finally, AuCePt PHNs-LA was stored at 4°C for subsequent experiments. SOD-like activity of AuCePt PHNs measurement The SOD-like activity of AuCePt PHNs was evaluated with a total superoxide dismutase assay kit (S0109, Beyotime Biotechnology, Shanghai, China). •O 2 − , generated by the reaction system of xanthine (X) and xanthine oxidase (XO), can reduce NBT to blue formazan, which has the strongest absorbance at 560 nm. Since SOD can scavenge •O 2 − , the SOD-like activity of AuCePt PHNs is inversely proportional to the production of blue formazan. Kinetic measurement: AuCePt PHNs (10 µL, 40 µg·mL − 1 ) or deionized water were mixed with the NBT working solution. The absorbance of the mixed solution at 560 nm was monitored for 30 min using a microplate reader [ 26 ]. End-point detection: Different concentrations of Au PHNs, AuCe PHNs, AuPt PHNs and AuCePt PHNs were mixed with the NBT working solution. After incubation at 37 ℃ for 30 min, the absorbance at 560 nm of the mixture was detected using a microplate reader. CAT-like activity of AuCePt PHNs measurement First, the CAT-like activity of AuCePt PHNs was evaluated by monitoring the O 2 produced during H 2 O 2 decomposition. Briefly, AuCePt PHNs (40 µg·mL − 1 ) and H 2 O 2 (100 mM) were added to PBS (25 mL) at three different pH values (5.5, 6.5, and 7.4). Oxygen solubility (mg·L − 1 ) was measured within 300 s using a dissolved oxygen meter. In addition, the CAT-like activities of three similar nanomaterials (Au PHNs, AuCe PHNs and AuPt PHNs) and AuCePt PHNs were compared by measuring the dissolved oxygen yield under the same conditions. In the second method, the CAT-like activity of AuCePt PHNs was evaluated using a CAT assay kit (S0051, Beyotime Biotechnology, Shanghai, China). First, a standard curve was established by using different concentrations of H 2 O 2 (0, 0.625, 1.25, 2.5, and 3.75 mM). Then, AuCePt PHNs and three similar nanomaterials (Au PHNs, AuCe PHNs and AuPt PHNs) were mixed with the precoordinated CAT reaction system. Finally, the absorbance of each sample at 520 nm was detected by a microplate reader after incubation in the dark at 25°C for 20 min. The residual H 2 O 2 was calculated as follows [ 27 ]: Residual H 2 O 2 = (A 520 -b)/K Detection of exogenous and endogenous ROS-scavenging activity The intracellular ROS level was monitored by using an ROS assay kit (C1300-1, Applygen Technologies, Beijing, China). AML-12 cells (5 × 10 6 cells·well − 1 ) were seeded in 6-well plates for 24 h, and then these cells were treated with the following substances: 1) saline, 2) AuCePt PHNs (2 µg·mL − 1 ), 3) H 2 O 2 (500 µM), and 4) AuCePt PHNs (2 µg·mL − 1 ) + H 2 O 2 (500 µM). Moreover, after incubation with free fatty acids (FFAs) mixture (1 mM, at a 2:1 ratio of oleate/palmitate) for 20 h, AML-12 cells were treated with 5) saline and 6) AuCePt PHNs (2 µg·mL − 1 ). After elution, these cells were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 µM) at 37°C for 30 min. Fluorescence images were recorded with a fluorescence microscope (Nikon ECLIPSE 80i, Japan), and the fluorescence intensity was measured with a Cary ECLIPSE fluorescence spectrophotometer (excitation: 502 nm, emission: 530 nm) [ 28 ]. Preparation of DSF-loaded AuCePt PHNs DSF (100 mg) was dissolved in dimethyl sulfoxide (DMSO, 10 mL). Subsequently, AuCePt PHNs (80 mg) were added to the DSF solution and stirred for 24 h in a darkroom. The mixture was centrifuged (8000 rpm × 20 min) and washed with DMSO 3 times to obtain AuCePt PHNs@DSF. Effect of pH value on DSF release DSF concentrations were analyzed by HPLC, and standard curves were established as previously reported [ 29 ]. To explore the drug release efficiency and pH dependence, AuCePt PHNs@DSF powder was dissolved in different pH (5.0 and 7.4) media. The supernatant was collected by centrifugation at 0.5, 1, 3, 6, 12, 24, and 48 h, and the same volume of fresh release medium was supplemented immediately. The concentration of DSF in the supernatant was analyzed by HPLC. DSF loading and entrapment rate of AuCePt PHNs The drug loading and encapsulation rate of AuCePt PHNs were calculated by the following equation: Cell culture, cytotoxicity test and construction of IR cell model AML-12, HepG-2, and HEK-293T cells were cultured in DMEM containing 10% fatal bovine serun (FBS) and 1% penicillin‒streptomycin at 37 ℃ and 5% CO 2 . For the cytotoxicity test, AML-12 cells (5 × 10 4 cells·well − 1 ) were plated in 96-well plates and treated with AuCePt PHNs (0, 10, 20, 50, 100 and 500 µg·mL − 1 ) or DSF (0, 2, 4, 6, 8 and 10 µM) for 3, 6, 12, 24, 48 and 72 h. Cell viability was measured using CCK-8 (Beyotime, Shanghai, China) [ 30 ]. To establish a lipid metabolism disorder or IR cell model, cells were incubated with FFAs mixture at 37°C for 20 h. For insulin signaling studies, cells were stimulated by insulin for 30 min. Flow cytometry For detection of apoptosis, AML-12 cells were labeled with annexin V and PI (Eliret Biotechnology Co., Ltd), as previously described [ 31 ]. Cells were acquired using multicolor flow cytometry (Beckman counter, DxFlex), and the fluorescence-activated cell sorting (FACS) data were analyzed using FlowJo (V10) software. Glucose uptake assay AML-12 IR cells treated with FFAs mixture were cultured in DMEM containing 1) control, 2) IR cells, 3) DSF (8 µM), 4) AuCePt PHNs-LA (2 µg·mL − 1 ), 5) DSF + AuCePt PHNs-LA (2 µg·mL − 1 ) and 6) AuCePt PHNs-LA@DSF (2 µg mL − 1 ) for 12 h. Cells were then collected and cultured in glucose-free DMEM for 30 min, and 2-NDBG (200 µM) was added for another 20 min. After washing three times, fluorescent images of intracellular 2-NBDG were obtained by a fluorescence microscope (Nikon ECLIPSE 80i), and the fluorescence intensity was measured at 540 nm by a fluorescence spectrophotometer [ 28 ]. Western blots Western blots were performed as previously reported [ 32 ]. Primary antibodies included anti-AKT/anti-phospho-AKT (9272S/4060S, Cell Signaling Technology), anti-IRS-1/anti-phospho-IRS-1 (2382S, Cell Signaling Technology), anti-PEPCK (sc-130,388, Santa Cruz), anti-FOXO-1 (2880S, Cell Signaling Technology), and anti-β-actin (17AV0410, ZSGB-BIO Inc.). Secondary antibodies (Multisciences Biotech, Hangzhou, China) were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies. Fluorescence imaging To verify the targeting of AuCePt PHNs-LA in vivo, fluorescence images were obtained. Briefly, AuCePt PHNs-LA (1 mL, 1 mg·mL − 1 ) was mixed with Cy7.5 (0.3 mL, 0.5 mg·mL − 1 ) to form AuCePt [email protected] . The mixture was collected by centrifugation, washed twice with PBS, and redissolved in PBS solution (200 µg·mL − 1 ). WT mice were given AuCePt [email protected] (200 µL) via tail vein injection. Fluorescence imaging was performed by a multispectral fluorescence system (visque in vivo smart, viewers, Korea). Animals and treatments To establish a diet-induced IR animal model, eight-week-old male C57BL/6J (WT) mice were fed a high-fat diet (HFD, 60 kcal% fat, D12492, Research Diets, New Brunswick, NJ) for 12 weeks. Subsequently, mice were randomly divided into four groups, and the following substances were given once every other day by tail vein for 16 days: 1) HFD (saline, 0.2 mL), 2) DSF (3 mg·kg − 1 , 0.2 mL), 3) AuCePt PHNs-LA (3 mg·kg − 1 , 0.2 mL), and 4) AuCePt PHNs-LA@DSF (3 mg·kg − 1 , 0.2 mL). For the intervention study in an animal model of diabetes, 8-week-old ob/ob mice (Jicui Yaokang Biotechnology, Jiangsu, China) were fed a standard diet (SD) for 2 weeks. Subsequently, mice were divided into two groups. and the following substances were given once every other day for 16 days: 1) SD (saline, 0.2 mL), 2) AuCePt PHNs-LA@DSF (3 mg·kg − 1 , 0.2 mL). Body weight was continuously monitored for 16 days. After the experiment, the mice were killed, and the tissues were stored at -160°C for subsequent experiments. The animal experiments were approved by the animal research committee of Chongqing Medical University and in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Indirect calorimetry and biochemical parameter analyses Indirect calorimetry was performed in a Comprehensive Lab Animal Monitoring System (MM-100; CWE, Ardmore, PA, USA). Body weight, heat and rectal temperature were measured at the indicated duration. The RER was calculated by 24-h VO 2 and 24-h VCO 2 as described previously [ 32 ]. Blood creatinine (Cre), urea nitrogen (BUN), creatine kinase (CK), hemoglobin (HGB), serum ALT and AST were determined by an automated biochemistry analyzer (ADVIA Chemistry XPT, Siemens Healthcare DiagnosticsInc, USA), as previously reported [ 33 ]. GTT and ITT tests For glucose tolerance test (GTT), mice were fasted overnight for 12 h and intraperitoneally injected with 20% glucose solution (7.5 µL·g − 1 ). Then, the concentration of glucose solution was adjusted so that the injection volume of each mouse was equal. For insulin tolerance test (ITT), mice were intraperitoneally injected with insulin (0.75 U·kg − 1 , Novolin) after fasting for 4 h, and blood glucose was measured at the designated time points as previously reported [ 34 , 35 ]. Histological examination Formalin-fixed livers from mice were processed, and paraffin sections (5 µm) were stained with H&E. Frozen liver sections were stained with 0.15% Oil Red O according to standard procedures as previously reported [ 36 ]. Statistical analysis Data are expressed as the mean ± standard error of mean or standard deviation, and statistical analyses were performed using SPSS 20.0 software (Chicago, IL, USA). Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test or unpaired Student’s t test. When p < 0.05, statistically significant differences were considered. Results and discussion Synthesis and characterization of AuCePt PHNs AuCePt PHNs were synthesized by sacrificial galvanic replacement of Co nanoparticles in the presence of HAuCl 4 , K 2 PtCl 6 and Ce(NO 3 ) 3 . Transmission electron microscopy (TEM) showed that AuCePt PHNs were spherical particles with a porous surface, hollow structure and average diameter of ~ 60 nm (Fig. 1 A), which is conducive to its passing through the hepatic sinusoid with a pore diameter of 50–180 nm of endothelial cells to reach hepatocytes or hepatic stellate cells [ 37 – 40 ]. Dynamic light scattering (DLS) experiments confirmed that the hydrodynamic size of AuCePt PHNs was ~ 66 nm (Fig. 1 E). The hydrodynamic diameter measured by DLS was slightly larger than that measured by TEM, which can be assigned to the fact that the size measured by DLS consisted of the diameters of both AuCePt PHNs and the hydration layer. The elemental mapping and energy dispersive X-ray spectroscopy (EDS) results showed that AuCePt PHNs were mainly composed of Au, Ce and Pt elements (Fig. 1 B and Figure S1 ). X-ray photoelectron spectroscopy (XPS) spectra of AuCePt PHNs also showed the characteristic peaks of Au, Ce and Pt elements (Fig. 1 C). Furthermore, the high-resolution XPS spectra fitted the characteristic peaks of each element in AuCePt PHNs (Figure S2), which showed Au containing 92.62% Au 0 , Ce containing 28.69% Ce 3+ and 71.31% Ce 4+ , and Pt containing 85.35% Pt 0 . High-resolution TEM was used to characterize the lattice spacing of the prepared materials. As shown in Figure S3A, the lattice spacing of AuCePt PHNs includes d [111] = 0.23 nm and d [200] = 0.20 nm, indicating the high crystallinity of AuCePt PHNs. The X-ray diffraction pattern (XRD) revealed the crystal planes of AuCePt PHNs, including the (111), (200), (220), (311) and (222) crystal planes, corresponding to 2θ values of 38.3°, 44.7°, 64.9°, 77.7° and 81.7°, respectively (Figure S3B). Compared with the standard cards (Au//PDF # 04-0784, Ce//PDF # 31–0325 and Pt//PDF # 04-0802) and XRD pattern of Au PHNs, the diffraction peaks of AuCePt PHNs were slightly shifted, revealing that the Au, Ce and Pt species formed a homogeneous and single-phase alloy structure [ 41 ]. The pore structure of AuCePt PHNs was analyzed by N 2 adsorption experiments (Fig. 1 D). The results showed that AuCePt PHNs had two main pore sizes of 3.5 nm and 6.1 nm. Moreover, the high-resolution TEM image also clearly showed a pore with a diameter of approximately 3.2 nm (Figure S3A, red arrow), which is mutually confirmed with the results of the N 2 adsorption experiment. Preparation and characterization of AuCePt PHNs-LA Because LA can specifically bind asialoglycoprotein receptor (ASGPR) on the surface of the hepatocyte membrane [ 42 ], AuCePt PHNs were modified with LA to target hepatocytes. L-cys with sulfhydryl and amino groups was used to link AuCePt PHNs and LA via Au-S and amide bonds. To analyze this process, the morphological changes of AuCePt PHNs were first characterized by TEM. As shown in Fig. 1 E, after being modified by L-cys, the surface of AuCePt PHNs clearly showed a transparent film. In addition, the transparent film became thicker with further connection of LA. The DLS results also showed that the diameter of AuCePt PHNs increased with the stepwise modification of L-cys and LA. Zeta-potential results (Figure S4) showed that the surface potential of AuCePt PHNs decreases from ~ -15 to ~ -20 mV and then to ~ -33 mV after the modification of L-cys and LA. These results confirmed the successful grafting of L-cys and LA. A previous study proved that the electrostatic repulsion of nanoparticles with zeta potentials greater than 20 mV or less than − 20 mV was sufficient to maintain the stability of nanoparticles in solvents [ 43 ]. Thus, AuCePt PHNs-LA with an ~ -33 mV surface charge may have good stability and dispersion in the circulating system. As expected, the diameter of AuCePt PHNs remained stable after being stored in PBS or serum for 1 month, indicating good stability and dispersion of AuCePt PHNs under physiological conditions (Figure S5). Next, 13 C nuclear magnetic resonance ( 13 C-NMR) was used to further confirm the successful modification of LA on AuCePt PHNs. Because AuCePt PHNs did not contain elemental carbon, there was no signal peak in the scanning spectrum of AuCePt PHNs (Fig. 1 F). After AuCePt PHNs were linked with L-cys and LA, signal peaks appeared at 24.7899, 25.2058, 42.6966 ~ 103.4555, 172.3389 and 176.5229 ppm. According to the report [ 44 ] and the 13 C-NMR scanning spectrum of L-cys and LA, the peaks at 61.0013–103.4555 ppm belong to LA, and the peaks at 24.7899, 55.895, 172.3389 ppm belong to L-cys, which confirmed that we successfully obtained the novel AuCePt PHNs-LA compounds. In addition, the changes in the functional groups of AuCePt PHNs, L-cys, and LA before and after connection were analyzed by Fourier transform infrared spectroscopy (FTIR). As shown in Fig. 1 G, L-cys had S-H stretching tensile vibration (blue frame) at 2550–2750 cm − 1 , but the peak disappeared after L-cys combined with AuCePt PHNs. This result indicated that the sulfhydryl of L-cys formed a more stable Au-S bond with AuCePt PHNs [ 45 ], and AuCePt PHNsPHN-L-cys was prepared. In addition, the vibration characteristic peak of C = O moved from 1741.03 cm − 1 to 1634.29 cm − 1 (red frame), which indicated that the -COOH of LA had successfully condensed with -NH 2 to form an amide bond [ 46 ] and proved that LA was modified on the surface of AuCePt PHNs-L-cys. Therefore, L-cys connects AuCePt PHNs and LA through gold sulfur bonds and amide bonds. Enzyme-like activity of AuCePt PHNs SOD catalyzes •O 2 − to generate H 2 O 2 and O 2 , which is the initial step of the scavenging ROS (Fig. 2 A). To evaluate the SOD-like activity of AuCePt PHNs, their performance in eliminating •O 2 − produced by xanthine (X) and xanthine oxidase (XO) reaction systems was monitored. The kinetic curve of absorbance at 560 nm in Fig. 2 B shows that the absorbance rises very slowly in the presence of AuCePt PHNs. This is because AuCePt PHNs can efficiently eliminate •O 2 − and thus inhibit the reduction of NBT to blue methadone, indicating that AuCePt PHNs have excellent SOD-like activity. In addition, by comparing the SOD-like activity of AuCePt PHNs with three other similar nanomaterials (AuCe PHNs, AuPt PHNs and Au PHNs), AuCePt PHNs have the highest SOD-like activity at any concentration (Fig. 2 C). H 2 O 2 is not only the product of •O 2 − but also another important ROS. It can be catalyzed by CAT to generate O 2 and H 2 O, which is the second key step in the ROS removal process (Fig. 2 D). Thus, the CAT-like activity of AuCePt PHNs was evaluated by monitoring the generation of O 2 . As shown in Fig. 2 E, AuCePt PHNs could efficiently catalyze H 2 O 2 to generate O 2 , and their CAT-like activity was the highest under neutral pH conditions. In addition, the O 2 generation rate increases with increasing AuCePt PHNs concentration. The oxygen production efficiency proved that the CAT-like activity of AuCePt PHNs was the highest among all nanomaterials (Figure S6A and B). Figure 2 F shows that the residual amount of H 2 O 2 in the AuCePt PHNs group was significantly lower than that in the Au PHNs, AuPt PHNs or AuCe PHNs groups, indicating that the AuCePt PHNs have the strongest enzyme activity for catalyzing H 2 O 2 decomposition. These results indicated that AuCePt PHNs present both SOD and CAT-like activities and can catalyze cascade enzyme reactions to eliminate ROS (•O 2 − --- H 2 O 2 --- O 2 + H 2 O). The outstanding enzymatic activity of AuCePt PHNs can be attributed to the following aspects: (1) •O 2 − readily captures protons from water, forming HO 2 • and OH − . The adsorption of HO 2 • on the Au(111) and Pt(111) planes is a highly exothermic process, and the activation energy barriers of the Au(111) and Pt(111) planes are extremely low. Once HO 2 • is adsorbed on the surface, Au and Pt exert SOD-like activity, converting HO 2 • into O 2 and H 2 O 2 [47] . (2) Under alkaline conditions, the Au(111) and Pt(111) planes can pre-adsorb OH, which serves as the active site to initiate the acid-like decomposition of H 2 O 2 , and can promote the conversion of H 2 O 2 to H 2 O and O 2 [18] . (3) Cerium nanomaterials have a redox pair that can cycle between the + 3 and + 4 states of oxygen vacancy sites, providing excellent SOD/CAT-like activity [ 19 ]. Furthermore, Fig. 2 (C and F) confirm our hypothesis that the combination of the three components would increase the enzyme-like activity of the AuCePt PHNs compared to that of single-component or two-component nanozymes. To investigate the ability of AuCePt PHNs to scavenge ROS at the cellular level, DCFH-DA was used as a fluorescent probe. As shown in Fig. 2 G and Figure S7 (A and B), treatment with AuCePt PHNs did not lead to significant changes in ROS levels in normal cells. After H 2 O 2 treatment, the fluorescence intensity was significantly increased, indicating that intracellular ROS levels were increased. However, the fluorescence was significantly reduced with the addition of AuCePt PHNs. These results suggested that AuCePt PHNs could scavenge exogenous ROS. To further explore the activity of AuCePt PHNs in scavenging endogenous ROS. Alpha mouse liver (AML-12) cells were treated with FFAs mixture to fabricate the IR model, which resulted in a significant increase in ROS levels (compared with normal cells). When AuCePt PHNs were added, the ROS level was significantly reduced, indicating that AuCePt PHNs effectively eliminated endogenous ROS. According to literature reports [ 26 ], since SOD and CAT are distributed in different organelles (Figure S8), the production and degradation of H 2 O 2 under natural conditions need to be transported between organelles. However, AuCePt PHNs have excellent SOD- and CAT-like activities simultaneously, which can catalyze cascade reactions, thus saving the transport process of H 2 O 2 between organelles. It is thus clear that the cascade response activity of AuCePt PHNs to ROS may be very helpful to improve the scavenging efficiency of ROS. Loading capacity and release efficiency of AuCePt PHNs for DSF In the above experiments, it has been confirmed that AuCePt PHNs have a porous and hollow physical structure. The literature has reported that this special physical structure endows nanoparticles with a strong load capacity [ 48 ]. According to the fitting linear equations of DSF detected by high-performance liquid chromatography (HPLC) (Figure S9) and the calculation equation in section 2.8, the loading rate of AuCePt PHNs for DSF was 37.3% ± 2.9, and the entrapment rate was 47.8% ± 5.8. Next, to analyze the release efficiency of AuCePt PHNs for DSF at different pH values, the cumulative release rate of DSF in 0–48 h was measured. Figure 3 A shows that the release of DSF from AuCePt PHNs is mainly concentrated within 24 h and has a significant pH dependence. When the pH of the dissolution medium was 7.4, the release efficiency in 48 h was only 36.98%, while when the pH was 5.5, the release efficiency reached approximately 74.76%. The pH dependence can reduce the nonspecific release of AuCePt PHNs to drugs in peripheral blood (pH = 7.35–7.45), while some organelles with lower pH (such as lysosomes) can promote the release of DSF from AuCePt PHNs [ 49 ]. Cytotoxicity and intracellular distribution of AuCePt PHNs To investigate the cytotoxicity of AuCePt PHNs and DSF, we first used Cell Counting Kit-8 (CCK-8) in AML-12 cells (Figure S10A and B). The results showed no significant cytotoxicity when the concentrations of AuCePt PHNs and DSF were less than 100 µg/mL and 8 µM, respectively. In addition, flow cytometric analysis further confirmed that there was no significant evidence of cell apoptosis in AML-12 cells after treatment with 100 µg/mL AuCePt PHNs or 8 µM DSF (Figure S11A and B). Figure 3 B clearly shows that AuCePt PHNs-LA was engulfed by cells through endocytosis and was encapsulated in endosomes. Importantly, the membrane and nuclear structures of the AuCePt PHNs-LA-treated AML-12 cells were clear and complete, which also proved that AuCePt PHNs-LA has good histocompatibility. In addition, we found that after AuCePt PHNs-LA was endocytosed, these endocytic particles were uniformly dispersed, which effectively avoided aggregation-induced cytotoxicity. To study the intracellular distribution of AuCePt PHNs-LA, AML-12 cells were treated with LysoTracker (red) and AuCePt PHNs-LA loaded with coumarin 6 (AuCePt PHNs-LA@cou6, green), and fluorescence colocalization analysis was performed at different time points. The results showed that the longer the treatment time of AuCePt PHNs-LA@cou6 was, the larger the yellow area (colocalization area) in cells, which peaked at 6 h (Fig. 3 C-F). This phenomenon indicated that the amount of AuCePt PHNs-LA entering the lysosome was the largest after incubation for 6 h. However, when the cells were incubated for 12 h, the green fluorescence and red fluorescence were obviously separated, indicating that some AuCePt PHNs-LA had escaped from the lysosome. The results of intracellular distribution and fluorescence colocalization indicated that AuCePt PHNs-LA could enter the lysosome so that DSF was effectively released in the acidic environment and could escape from the lysosome to play a therapeutic role in the cytoplasm. Effects of AuCePt PHNs-LA@DSF on intracellular IR The researches of Prof. Mailloux and Prof. Sun proved that DSF can clear ROS and reduce OS [ 50 , 51 ]. Furthermore, the above experiments confirmed that AuCePt PHNs-LA can reduce the endogenous and exogenous ROS. Therefore, we speculate that AuCePt PHNs-LA and DSF can jointly improve IR by reducing OS. To explore the effect of AuCePt PHNs-LA@DSF on cell-level IR, we conducted a 2-NBDG uptake experiment in AML-12 IR cells. Cell fluorescence imaging showed that the fluorescence intensity of the four treatment groups (DSF, AuCePt PHNs-LA, and AuCePt PHNs-LA@DSF) was higher than that of control group (Fig. 4 A). This finding suggested that they can improve glucose uptake in IR cells. Among them, AuCePt PHNs-LA@DSF group had the highest glucose uptake rate. Similar results were obtained by flow cytometry analysis (Fig. 4 B). In addition, the glycogen staining results showed that the glycogen content in IR cells treated with AuCePt PHNs-LA and DSF was significantly increased, while the cell glycogen content was the highest after AuCePt PHNs-LA@DSF treatment (Fig. 4 C and D). Therefore, the AuCePt PHNs-LA@DSF can efficiently promote glucose uptake and glycogen synthesis in IR cells, thereby improving glucose metabolism. Influence of AuCePt PHNs-LA@DSF on gluconeogenesis and insulin signaling molecules in IR hepatocytes It has been well documented that OS promote the occurrence and development of IR through insulin signaling pathway and gluconeogenesis signaling pathway [ 41 , 42 ]. Therefore, we next explored the influence of AuCePt PHNs-LA@DSF on the phosphorylation and expression of key molecules in these two classic pathways, including IRS-1, AKT (a serine/threonine kinase and a versatile node in insulin signal transduction) [ 52 ], FOXO-1 (an important substrate molecule downstream of IRS-1/AKT, mediating gluconeogenesis) [ 53 ], and PEPCK (a rate-limiting enzyme in the gluconeogenesis pathway) [ 54 ]. Firstly, we investigated the effects of different concentrations of Au and DSF on AKT phosphorylation. As shown in Fig. 4 E-H, AuCePt PHNs-LA or DSF improved AKT phosphorylation levels in IR AML-12 cells in a dose-dependent manner. Secondly, by comparing the effects of different nanomaterials on the AKT phosphorylation level, it could be seen that AuCePt PHNs had the highest regulation efficiency (Figure S12). Significantly, treatment with DSF, AuCePt PHNs-LA, AuCePt PHNs-LA@DSF resulted in increased IRS-1 and AKT phosphorylation levels, and decreased FOXO1 and PEPCK expression in IR AML-12 cells. Especially, the effect of AuCePt PHNs-LA@DSF is the best (Fig. 4 I ~ M). These results indicated that AuCePt PHNs-LA@DSF can improve IR by regulating the insulin signaling pathway and the gluconeogenesis signaling pathway. Targeting efficiency, biodistribution and toxicity of AuCePt PHNs-LA in vivo To investigate whether the LA-modified AuCePt PHNs can promote the phagocytosis of hepatocytes, we used doxorubicin (DOX) to label AuCePt PHNs and AuCePt PHNs-LA to construct fluorescent probes. AML-12 or HEK293T cells were treated with AuCePt PHNs or AuCePt PHNs-LA as indicated in the Methods. We found that the fluorescence signal in AML-12 cells treated with AuCePt PHNs-LA@DOX was significantly higher than that in AuCePt PHNs@DOX-treated AML-12 cells, indicating that LA modification improved the phagocytosis efficiency of AuCePt PHNs in AML-12 cells (Fig. 5 A). In addition, in AuCePt PHNs-LA@DOX-treated cells, the fluorescence signal in HEK293T cells was significantly weaker than that in AML-12 cells, indicating that LA modification promoted the ASGPR-mediated endocytosis of AuCePt PHNs-LA, which is highly expressed on the surface of hepatocytes. Based on LA functional modification and the pH dependence of drug release, AuCePt PHNs-LA could efficiently target the liver to release DSF, thereby reducing the nonspecific distribution and toxicity of nanomaterials and drugs. To understand the biodistribution of AuCePt PHNs-LA in vivo, WT mice were injected with Cy7.5-labeled AuCePt PHNs or AuCePt PHNs-LA via the tail vein. In vivo fluorescence imaging showed that AuCePt [email protected] accumulated more in the liver than did AuCePt [email protected] after 3h of injection, suggesting that AuCePt PHNs-LA were more targeted (Fig. 5 B). Consistently, organ fluorescence imaging also confirmed this conclusion (Fig. 5 C and Figure S13). We continued to explore the distribution of AuCePt PHNs-LA in organs at different time points. The fluorescence of the liver decreased gradually over time. Importantly, the liver still retained a strong fluorescence signal after 48 h, which indicated that AuCePt PHNs-LA can stay in the liver for a long time to exert its enzyme-like activity. The fluorescence intensity in the kidney increased, suggesting that the material may be excreted through the kidney. Next, to verify the toxicity of DSF and AuCePt PHNs-LA in vivo, we analyzed routine blood and biochemical indicators in mice. Compared with HFD group, DSF, AuCePt PHNs-LA, and AuCePt PHNs-LA@DSF treatment did not significantly affect various indicators in the mice (Table S1 ). In addition, there was no significant change in H&E staining of various tissues or organs (Figure S14). These results indicated that AuCePt PHNs-LA@DSF did not cause cardiac or renal dysfunction or systemic functional disorders in mice. Therefore, AuCePt PHNs-LA@DSF has good biosecurity. Effect of AuCePt PHNs-LA@DSF on energy expenditure in vivo To establish HFD-induced obese animal models, WT mice were fed a HFD for 12 weeks and treated with DSF, AuCePt PHNs-LA, or AuCePt PHNs-LA@DSF for 16 days (Fig. 5 D). Compared with the HFD group, the mice in the treatment groups were significantly smaller body weight and in size (Fig. 5 E and F). In particular, the weight loss was most obvious in the AuCePt PHNs-LA@DSF-treated group. In addition, the morphological, H&E staining and oil-red O staining images of the liver showed that lipid deposition was significantly reduced in the treatment groups (Fig. 5 G and Figure S15 ~ 16). Similarly, among several treatments, the AuCePt PHNs-LA@DSF treatment was the most effective at reducing lipid deposition. One of the important reasons for obesity is an imbalance in energy metabolism, that is, an increase in energy storage and a decrease in energy consumption. Therefore, we measured the energy expenditure in each treatment group with metabolic cages. We found that oxygen consumption (VO 2 ) (Fig. 5 H) and carbon dioxide (VCO 2 ) production (Figure S17A and B) increased significantly in the three treatment groups, indicating a higher metabolic rate. The respiratory exchange ratio (RER) also increased to varying degrees, especially in the AuCePt PHNsPHN-LA@DSF group (Fig. 5 I). A higher RER meant a higher utilization rate of carbohydrates, implying that IR was alleviated. Moreover, the whole-body energy expenditure and rectal temperature also increased significantly (Figure S17C and D). Overall, these data indicated that AuCePt PHNs-LA@DSF treatment significantly reduced body weight and liver lipid accumulation and increased overall energy consumption. Moreover, our data suggested that AuCePt PHNs-LA and DSF exhibit combined therapeutic effects. In addition, these results proved that the increase in energy consumption is the main cause of weight loss in the AuCePt PHNs-LA@DSF treatment group. Effects of AuCePt PHNs-LA@DSF on glucose metabolism and IR in vivo To evaluate the effect of AuCePt PHNs-LA@DSF on insulin sensitivity in vivo, WT mice were fed a HFD for 12 weeks to construct an IR model and then treated with DSF, AuCePt PHNs-LA, DSF + AuCePt PHNs-LA or AuCePt PHNs-LA@DSF for 16 days via the tail vein (Fig. 6 A). As shown in Fig. 6 B ~ C, fasting blood glucose and insulin levels in the three treatment groups were markedly reduced. In particular, the fasting blood glucose and insulin levels of AuCePt PHNs-LA@DSF group mice were very close to those of the control group mice. Moreover, during the GTT and ITT experiments, the blood glucose level and the area under the glucose curve (AUC) at each time point in the three treatment groups significantly decreased, while those in the AuCePt PHNs-LA@DSF-treated group were the lowest (Fig. 6 D ~ G). To further explore the effect of AuCePt PHNs-LA@DSF on insulin sensitivity in the liver at the molecular level, we measured the level of phosphorylation or expression of insulin and gluconeogenesis signaling molecules extracted from liver tissues. Compared with the control group, the phosphorylation levels of IRS-1 and AKT were significantly increased in the liver, while the levels of PEPCK and FOXO1 proteins were significantly decreased, suggesting that insulin sensitivity was improved in the liver, especially in AuCePt PHNs-LA@DSF-treated mice (Fig. 6 H ~ L). Chronic low-grade inflammation leads to liver steatosis, IR and obesity. Thus, the effect of AuCePt PHNs-LA@DSF on OS and inflammation in the liver was analyzed. The ROS-scavenging efficiency of each treatment group was measured using DCFH-DA as the fluorescent probe. We found that AuCePt PHNs-LA and DSF treatment significantly reduced ROS levels in the livers of HFD-fed C57BL/6J mice (Figure S18). The fluorescence intensity of the AuCePt PHNs-LA@DSF-treated group was the weakest, indicating that its antioxidant capacity was the strongest. In addition, we investigated the effects of AuCePt PHNs-LA@DSF on inflammatory factors and liver enzymes in vivo. As shown in Fig. 6 M ~ P, these three treatment groups all exhibited significant decreases in serum TNF-α and IL-6 levels and significant improvements in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. These changes were most obvious in the AuCePt PHNs-LA@DSF-treated group, suggesting that the combination of DSF and AuCePt PHNs-LA had the strongest anti-inflammatory effect and ameliorated liver injury. Efficacy verification of AuCePt PHNs-LA@DSF on ob/ob mice Ob/ob mice is a classic model of genetic obesity, accompanied by liver steatosis [ 55 ]. Therefore, to evaluate the effects of AuCePt PHNs-LA@DSF treatment on IR, obesity and fatty liver, ob/ob mice were treated with AuCePt PHNs-LA@DSF for 16 days via the tail vein (Fig. 7 A). The results showed that AuCePt PHNs-LA@DSF treatment significantly reduced body weight (Fig. 7 B ~ C, and Figure S19A), fasting blood glucose (Fig. 7 D), fasting insulin (Figure S19B), and liver lipid deposition (Fig. 7 E and Figure S19C). In addition, the glucose tolerance and insulin tolerance improved significantly (Fig. 7 F ~ I). Moreover, ROS levels and inflammation in the livers of ob/ob mice were alleviated (Figure S19D). Western blot analysis revealed that compared with the SD-fed control group, the protein expression of PEPCK and FOXO1 in AuCePt PHNs-LA@DSF-treated ob/ob mice was significantly inhibited, while the phosphorylation of IRS-1 and AKT were significantly increased (Fig. 7 J-N). These data indicated that AuCePt PHNs-LA@DSF treatment efficiently inhibited gluconeogenesis and improved insulin sensitivity in ob/ob mice. Conclusion In summary, we successfully synthesized a novel porous hollow cascade nanozymes, AuCePt PHNs, which were functionalized with LA and loaded with DSF to alleviate hepatic IR by reducing OS. AuCePt PHNs simultaneously had SOD/CAT-like activity and exhibited extremely strong loading and pH-dependent release capacity for DSF based on their porous and hollow structures, which effectively reduced the nonspecific release of DSF in the peripheral blood circulation. In vitro experiments proved that AuCePt PHNs-LA could improve glucose uptake and glycogen synthesis in AML-12 IR cells by scavenging endogenous and exogenous ROS, while this antioxidative effect was significantly enhanced after loading with DSF (AuCePt PHNs-LA@DSF). Studies in HFD-fed mice revealed that AuCePt PHNs-LA@DSF can significantly reduce blood glucose levels and improve liver lipid deposition, IR and obesity. Even in diabetic ob/ob mice, the above effects of AuCePt PHNs-LA@DSF were also obvious, suggesting that it has an excellent antidiabetic effect. In addition, compared with single administration, the better therapeutic efficacy of AuCePt PHNs-LA@DSF indicates that AuCePt PHNs-LA and DSF have combined effects in the field of antioxidant therapy. Therefore, this work illustrates that AuCePt PHNs-LA@DSF can improve insulin sensitivity in diabetic mice by modulating IRS-1/AKT and gluconeogenesis signaling pathways and provides a promising therapeutic strategy for OS-related obesity, fatty liver disease and diabetes. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution HW.S: Conceptualization, Methodology, Investigation, Formal analysis, Writing-Original Draft, Funding acquisition.YF.F: Resources, Investigation, Formal analysis, Writing-Original Draft. FF.L: Resources, Investigation, Writing - Review & Editing.WL.Z: Writing - Review & Editing. Y.Y: Resources.GY.Y: Conceptualization, Methodology, Supervision, Project administration, Writing - Review & Editing.ML.Y: Formal analysis, Writing - Review & Editing.L.L: Resources, Supervision, Project administration, Funding acquisition. Acknowledgments This work was financially supported by the National Natural Science Foundation for the Youth of China (No. 81902171), the National Natural Science Foundation of China (No. 82170816), and the Natural Science Foundation of Chongqing, China (cstc2020jcyj-msxmX0040). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Wang J, Ma J, Nie H, Zhang XJ, Zhang P, She ZG, Li H, Ji YX, Cai J. Hepatic Regulator of G Protein Signaling 5 Ameliorates Nonalcoholic Fatty Liver Disease by Suppressing Transforming Growth Factor Beta-Activated Kinase 1-c-Jun-N-Terminal Kinase/p38 Signaling. Hepatology. 2021;73:104–25. 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Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 26 Oct, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 18 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviews received at journal 13 Jul, 2024 Reviews received at journal 06 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers agreed at journal 30 Jun, 2024 Reviewers agreed at journal 27 Jun, 2024 Reviewers invited by journal 25 Jun, 2024 Editor assigned by journal 15 Jun, 2024 Submission checks completed at journal 15 Jun, 2024 First submitted to journal 14 Jun, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4580829","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320823483,"identity":"2b7e5b51-36ed-4316-a32b-b16bdf7d122a","order_by":0,"name":"Huawei Shen","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huawei","middleName":"","lastName":"Shen","suffix":""},{"id":320823484,"identity":"1d01834b-c3e9-42ba-93ac-9a9cd8c19f38","order_by":1,"name":"Yafei Fu","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"Fu","suffix":""},{"id":320823485,"identity":"9b55a99f-8328-4585-bf80-d059f8c62691","order_by":2,"name":"Feifei Liu","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Liu","suffix":""},{"id":320823486,"identity":"caa39ab7-e8d7-480d-b782-d8ed8b118a16","order_by":3,"name":"Wanliang Zhang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wanliang","middleName":"","lastName":"Zhang","suffix":""},{"id":320823487,"identity":"71aa60cf-009e-4361-a5c8-567d58d4cc62","order_by":4,"name":"Yin Yuan","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Yuan","suffix":""},{"id":320823488,"identity":"49c8165a-7e40-40c7-bdd3-e76c6a84daf2","order_by":5,"name":"Gangyi Yang","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gangyi","middleName":"","lastName":"Yang","suffix":""},{"id":320823489,"identity":"19ca297a-b000-4983-b8cf-b4857ea9b354","order_by":6,"name":"Mengliu Yang","email":"","orcid":"","institution":"Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengliu","middleName":"","lastName":"Yang","suffix":""},{"id":320823490,"identity":"ca7b49fa-0143-4946-9914-d8a4d1264688","order_by":7,"name":"Ling Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACPmbmhgMMDDZQLhsRWtiYGUFa0kjRwsDYAKQOk6KFnbHxcMGv8/J8184YMHwoO8zAP7uBsMMOz+y7bTjzdo4B44xzhxkk7hwgQgtvz+0EA6AWZt62wwwGEglEaTkH0fKXaC08Pw5AtDASb0tDMtAvaQUHe86l80jcIKCFn//w4c88f+zk+W4nb3zwo8xajn8GAS1gwNgGJA6AEQMPEepB4A9EyygYBaNgFIwCrAAAhDFCdj4Cd8sAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ling","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-14 09:02:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4580829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4580829/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02880-z","type":"published","date":"2024-10-26T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59497575,"identity":"555a2a25-3568-45ee-95de-acb8325e30ea","added_by":"auto","created_at":"2024-07-02 13:34:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":257693,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of AuCePt PHNs and AuCePt PHNs-LA. (\u003cstrong\u003eA\u003c/strong\u003e) TEM image of AuCePt PHNs. (\u003cstrong\u003eB\u003c/strong\u003e) Elemental mapping images of AuCePt PHNs. (\u003cstrong\u003eC\u003c/strong\u003e) XPS spectra of AuCePt PHNs. (\u003cstrong\u003eD\u003c/strong\u003e) N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherms (inset) and the corresponding DFT pore size distribution curves of AuCePt PHNs. (\u003cstrong\u003eE\u003c/strong\u003e) DLS and high-magnification TEM characterization of the particle size and morphology of AuCePt PHNs-LA. (\u003cstrong\u003eF\u003c/strong\u003e) \u003csup\u003e13\u003c/sup\u003eC-NMR spectroscopy and (\u003cstrong\u003eG\u003c/strong\u003e) FTIR spectroscopy of AuCePt PHNs modified by L-cys and LA.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/66b3120b20cc8cb2113105ca.png"},{"id":59497576,"identity":"24cae9d1-9f15-4a6d-bcd0-dbd7fd1734eb","added_by":"auto","created_at":"2024-07-02 13:34:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":424684,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the SOD and CAT-like activities of AuCePt PHNs.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Simulation diagram for the SOD-like activity of AuCePt PHNs. (\u003cstrong\u003eB\u003c/strong\u003e) Typical kinetic curves of A − A0 (560 nm) for monitoring the reduction of NBT with •O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e. (\u003cstrong\u003eC\u003c/strong\u003e) •O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e elimination efficiency of similar nanozymes with different concentrations. (\u003cstrong\u003eD\u003c/strong\u003e) Simulation diagram for the CAT-like activity of AuCePt PHNs. (\u003cstrong\u003eE\u003c/strong\u003e) Typical kinetic curves of AuCePt PHNs-mediated O\u003csub\u003e2\u003c/sub\u003e generation from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at different pH values. (\u003cstrong\u003eF\u003c/strong\u003e) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition efficiency of similar nanozymes with different concentrations. (\u003cstrong\u003eG\u003c/strong\u003e) Effects of AuCePt PHNs on ROS levels in AML-12 cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or FFAs.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/3aa42370bfeccb5e84de3d8d.png"},{"id":59497577,"identity":"f276e56d-f5a5-454b-947c-f47d4f12948e","added_by":"auto","created_at":"2024-07-02 13:34:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":650714,"visible":true,"origin":"","legend":"\u003cp\u003ePhysical properties and intracellular localization of AuCePt PHNs. (\u003cstrong\u003eA\u003c/strong\u003e) Efficiency-time curves of AuCePt PHNs for DSF release at different pH values. (\u003cstrong\u003eB\u003c/strong\u003e) TEM images of the intracellular distribution of AuCePt PHNs-LA. (\u003cstrong\u003eC-F\u003c/strong\u003e) Colocalization analysis for AuCePt PHNs (green) and lysosomes (red) at different time periods.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/cbedfb944bf9ac61e786c778.png"},{"id":59496958,"identity":"23b367b1-b1aa-42cb-9835-c2aca248e00e","added_by":"auto","created_at":"2024-07-02 13:26:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":906678,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AuCePt PHNs-LA@DSF on glucose metabolism and the expression of insulin signaling molecules in vitro. The control group is the IR cells model, which is prepared by AML-12 cells incubating with FFAs for 20 h. (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescent images for 2-NBDG uptake level. (\u003cstrong\u003eB\u003c/strong\u003e) Flow cytometry for 2-NBDG uptake level. (\u003cstrong\u003eC\u003c/strong\u003e) PAS staining for glycogen contents. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative analysis of glycogen contents. WB experiments: (\u003cstrong\u003eE~F\u003c/strong\u003e) Akt phosphorylation level in AuCePt PHNs-treated AML-12 IR cells. (\u003cstrong\u003eG~H\u003c/strong\u003e) Akt phosphorylation level in DSF-treated AML-12 IR cells. (\u003cstrong\u003eI~M\u003c/strong\u003e) The expression and phosphorylation of key proteins in insulin signaling pathway and gluconeogenesis signaling pathway in DSF, AuCePt PHNs-LA, and AuCePt PHNs-LA@DSF-treated AML-12 IR cells. Data are expressed as the mean ± standard deviation (n = 3 independent experiments). *p \u0026lt;0.05, **p \u0026lt;0.01, and ***p \u0026lt;0.001 vs control.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/c0eb71b9a59d772addeb2726.png"},{"id":59496955,"identity":"19b08586-b446-471f-8299-3b3f30b0c6cb","added_by":"auto","created_at":"2024-07-02 13:26:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1092147,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of AuCePt PHNs-LA for targeting the liver: (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescence emission spectroscopy and quantitative analysis of the endocytosis level of AuCePt PHNs by different cells before and after LA modification (\u003cstrong\u003eB\u003c/strong\u003e) In vivo imaging showing the distribution of AuCePt PHNs or AuCePt PHNs-LA in mice after tail vein injection. (\u003cstrong\u003eC\u003c/strong\u003e) Fluorescence images showing the distribution of AuCePt PHNs and AuCePt PHNs-LA in different organs after tail vein injection. Effects of AuCePt PHNs-LA@DSF on body weight and energy metabolism in HFD-fed C57BL/6J mice: (\u003cstrong\u003eD\u003c/strong\u003e) Experimental strategy for measuring energy expenditure. Cumulative body weight (\u003cstrong\u003eE\u003c/strong\u003e), representative photographs of the whole body (\u003cstrong\u003eF\u003c/strong\u003e), and HE staining of liver tissue sections (\u003cstrong\u003eG\u003c/strong\u003e) of mice subjected to different treatments (a. HFD, B. DSF, C. AuCePt PHNs-LA, and D. AuCePt PHNs-LA@DSF). (\u003cstrong\u003eH\u003c/strong\u003e) 24 h oxygen consumption. (\u003cstrong\u003eI\u003c/strong\u003e) Respiratory exchange ratio (RER: VCO\u003csub\u003e2\u003c/sub\u003e/VO\u003csub\u003e2\u003c/sub\u003e). Data are expressed as the mean ± standard deviation or standard error of mean (n = 4 ~ 6 mice for each group), * p \u0026lt;0.05, **p \u0026lt;0.01, ***p \u0026lt; 0.001 vs. HFD group.\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/aff3cc8cad5d0fb53cbe739c.png"},{"id":59496964,"identity":"5aa52041-8685-4f2e-8df3-b509060d1507","added_by":"auto","created_at":"2024-07-02 13:26:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":404469,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AuCePt PHNs-LA@DSF on glucose metabolism and insulin sensitivity in HFD-fed mice. (\u003cstrong\u003eA\u003c/strong\u003e) Experimental strategy for GTT and ITT. (\u003cstrong\u003eB\u003c/strong\u003e) Fasting blood glucose. (\u003cstrong\u003eC\u003c/strong\u003e) Fasting insulin. (\u003cstrong\u003eD\u003c/strong\u003e) and (\u003cstrong\u003eE\u003c/strong\u003e) Blood glucose and AUC\u003csub\u003eGTT \u003c/sub\u003eduring the GTT. (\u003cstrong\u003eF\u003c/strong\u003e) and (\u003cstrong\u003eG\u003c/strong\u003e) Blood glucose and AUC\u003csub\u003eITT \u003c/sub\u003eduring the ITT. (\u003cstrong\u003eH - L\u003c/strong\u003e) Protein expression of PEPCK and FOXO1 and phosphorylation levels of AKT and IRS-1. (\u003cstrong\u003eM\u003c/strong\u003e) Serum TNF-α levels. (\u003cstrong\u003eN\u003c/strong\u003e) Serum IL-6 levels. (\u003cstrong\u003eO\u003c/strong\u003e) Serum AST levels. (\u003cstrong\u003eP\u003c/strong\u003e) Serum ALT levels. Data are expressed as the mean ± standard deviation (n = 6 mice for each group), * p \u0026lt;0.05, **p \u0026lt;0.01 and ***p \u0026lt;0.001 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/73915e6f02587e3408a73530.png"},{"id":59496963,"identity":"d3952881-1448-4bad-9a1f-f797f39f4e04","added_by":"auto","created_at":"2024-07-02 13:26:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1892579,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AuCePt PHNs-LA@DSF on glucose metabolism and insulin sensitivity in ob/ob mice. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the experimental procedure. (\u003cstrong\u003eB\u003c/strong\u003e) Representative photographs of the whole body of the mice. (\u003cstrong\u003eC\u003c/strong\u003e) Body weight. (\u003cstrong\u003eD\u003c/strong\u003e) Fasting blood glucose. (\u003cstrong\u003eE\u003c/strong\u003e) Hepatic H\u0026amp;E staining. (\u003cstrong\u003eF\u003c/strong\u003e) and (\u003cstrong\u003eG\u003c/strong\u003e) Blood glucose and AUC\u003csub\u003eGTT \u003c/sub\u003eduring the GTT. (\u003cstrong\u003eH\u003c/strong\u003e) and (\u003cstrong\u003eI\u003c/strong\u003e) Blood glucose and AUC\u003csub\u003eITT \u003c/sub\u003eduring the ITT. (\u003cstrong\u003eJ~N\u003c/strong\u003e) Expression of PEPCK and FOXO1 proteins and phosphorylation levels of AKT and IRS-1. Data are expressed as the mean ± standard deviation (n = 4 ~ 6 mice for each group), **p \u0026lt;0.01, ***p \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/e7b4a582d35f82d1116b1155.png"},{"id":67681890,"identity":"5fda0d47-e853-48f2-95aa-535d427e3b9b","added_by":"auto","created_at":"2024-10-28 16:10:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8118994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/827bfc1e-5d75-4ed5-bdcd-00a26f413f75.pdf"},{"id":59496961,"identity":"c4811261-1795-4dab-bbdf-bbec4516efff","added_by":"auto","created_at":"2024-07-02 13:26:55","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5265000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eSchematic diagram of (A) the preparation of AuCePt PHNs-LA@DSF nanocomposites, and (B) the targeted improvement of hepatic IR by AuCePt PHNs-LA@DSF.\u003c/p\u003e","description":"","filename":"Scheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/2983a4d7ffa979a51d6a5bac.tif"},{"id":59496967,"identity":"490c9a78-afa0-4f61-a666-60bd4f58b6f1","added_by":"auto","created_at":"2024-07-02 13:26:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6535900,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4580829/v1/3655c249062f0931123584f7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"AuCePt porous hollow cascade nanozymes targeted delivery of disulfiram for alleviating hepatic insulin resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetabolic diseases such as obesity, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) are the most common metabolic diseases at present, with billions of patients worldwide, and the incidence rate is increasing rapidly. In China, the prevalence of NAFLD has exceeded 30%, and the prevalence of T2DM has exceeded 11.5%, which has become a major public health problem and a serious social medical burden [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The pathophysiological basis of the occurrence and development of these metabolic diseases is metabolic disorder and insulin resistance (IR) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, to combat metabolic diseases, it is necessary to use safe and effective drugs for a long time. Ideal drugs can not only improve metabolic disorders in vivo but also ameliorate insulin sensitivity and have better targeting and fewer side effects on metabolic tissues and organs. In the past 50 years, although extensive research has been carried out and some progress has been made, there is still no ideal drug for clinical application in the treatment of metabolic diseases. Therefore, it is necessary to adopt new approaches and drugs to restore systemic metabolic homeostasis to help cope with the increasingly serious public health crisis caused by metabolic diseases.\u003c/p\u003e \u003cp\u003eOxidative stress (OS) is one of the important reasons for the occurrence and development of IR [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which is caused by the imbalance between the production and clearance of reactive oxygen species (ROS) and reactive nitrogen species (RNs). Studies have shown that OS can activate and induce IκB kinase (IKK)/NF-кB and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) signaling pathways, interfere with the signal transduction of insulin receptors (InsR) and the phosphorylation of downstream signaling molecules such as insulin receptor substrate 1 (IRS-1) and protein kinase B (AKT), and inhibit the expression of glucose transporters, leading to glucose metabolism disorder and IR [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, one of the effective ways of improving IR to ameliorate antioxidant activity is by eliminating excessive ROS and reducing OS in vivo.\u003c/p\u003e \u003cp\u003eWith the development of nanotechnology, various nanomaterials have been developed and widely used in the diagnosis and treatment of diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Targeted delivery of drugs has become a hotspot in tumor research, which increases the concentration and efficiency of drugs in the lesion site and reduces the toxic and side effects caused by nonspecific distribution of drugs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nanozymes, another research hotspot in nanoscience, are an umbrella term for a class of nanomaterials with similar biological enzymatic activities [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. They can simulate the activity of one or more biological enzymes, such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT). For instance, Au nanomaterial can simulate the activities of POD, CAT, and SOD. More importantly, Au nanomaterial boasts high stability and biocompatibility and can be functionally modified via gold-ammonia bonds and gold-sulfur bond [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e adsorption affinity of the Pt (111) plane is higher than that of the Au (111) plane, which endows the Pt nanomaterial with higher catalytic activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Ce nanomaterials also have excellent SOD-, CAT-, and oxidase-like activities owing to the reversible conversion of their ionic state between Ce\u003csup\u003e3+\u003c/sup\u003e and Ce\u003csup\u003e4+\u003c/sup\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the catalytic activity and biological function of single-component nanomaterials are relatively limited [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, if the advantages of the three (Au/Ce/Pt) components can be combined, the catalytic activity and biological functions of nanozymes can be notably enhanced.\u003c/p\u003e \u003cp\u003eFor many years, disulfiram (DSF), which was originally found to be an inhibitor of nuclear factor-κB (NFκB), has been widely used to treat alcoholism. It has anti-inflammatory properties and can treat a variety of cancers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent study found that DSF treatment resulted in weight loss and improved insulin sensitivity and liver lipid accumulation in mice fed a high-fat diet (HFD) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Although there is a preliminary understanding of the role of DSF in metabolic regulation, previous studies have been relatively shallow observational studies. In particular, DSF lacks targeting to major metabolic organs, such as the liver. Therefore, the development of DSF vectors targeting the liver has important clinical application value for the treatment of metabolic disorders.\u003c/p\u003e \u003cp\u003eIn the current study, we constructed a novel nanozyme capable of cascading ROS scavenging, namely AuCePt porous hollow cascade nanozymes (AuCePt PHNs), which can simulate SOD/CAT-like activities to catalyze a cascade of enzyme reactions, thereby efficiently scavenging ROS and inhibiting OS. AuCePt PHNs were modified by lactobionic acid (LA) and loaded with DSF (AuCePt PHNs-LA@DSF) for targeted hepatocyte delivery and combined treatment of hepatic IR (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that AuCePt PHNs-LA@DSF could improve glucose uptake and glycogen synthesis of IR hepatocytes by activating the IRS-1/AKT signal pathway and inhibiting the forkhead box O1 (FOXO-1)/phosphoenolpyruvate carboxykinase (PEPCK) signal pathway. It was also found that the intravenous injection of AuCePt PHNs-LA@DSF reduced body weight and blood glucose levels and improved hepatic IR in HFD-fed mice and leptin-deficient (ob/ob) diabetic mice. In general, our research results revealed the beneficial role of AuCePt PHNs-LA@DSF in controlling blood glucose, improving hepatic insulin sensitivity and lipid deposition, and suggested its potential efficacy in the treatment of OS-related metabolic diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of AuCePt PHNs\u003c/h2\u003e \u003cp\u003eAuCePt PHNs were synthesized according to previously reported methods with some modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, Na\u003csub\u003e3\u003c/sub\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e solution (400 \u0026micro;L, 0.1 M) and NaBH\u003csub\u003e4\u003c/sub\u003e (400 \u0026micro;L, 1.0 M) were added to the preprepared dissolved oxygen-depleted ddH\u003csub\u003e2\u003c/sub\u003eO (100 mL) and stirred for 5 min. With the addition of cobalt chloride solution (100 \u0026micro;L, 0.5 M), the solution color rapidly changed from colorless to gray. Immediately, HAuCl\u003csub\u003e4\u003c/sub\u003e solution (200 \u0026micro;L, 0.1 M) was added dropwise. After stirring continued for 20 min, the nitrogen gas was stopped. When the color of the solution changed from gray‒red to dark blue, Ce(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e (100 \u0026micro;L, 0.1 M) and K\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e (100 \u0026micro;L, 0.1 M) were added into the mixture and continuously stirred for 30 min. Finally, AuCePt PHNs were collected by centrifugation and washed three times with deionized water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of AuCePt PHNs-LA\u003c/h2\u003e \u003cp\u003eBecause L-cysteine (L-Cys) has both sulfhydryl and amino groups, it was used as an intermediate molecule to connect AuCePt PHNs and LA through gold-sulfur bonds and amide bonds [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. As-prepared AuCePt PHNs (40 mg) were dispersed into a freshly prepared L-cys solution (10 mL, 1 mM). After vigorous stirring for 3 min, the mixture was allowed to stand at 4\u0026deg;C for 16 h. Next, the mixture was washed with deionized water three times to remove the excess L-cys ligands. LA (8 mg), EDC\u0026sdot;HCl (4.7 mg) and NHS (2.8 mg) were dissolved in 1 mL deionized water and stirred for 3 h. The above solution was mixed with AuCePt PHNs-L-cys (9 mL) and then stirred for 16 h at 4\u0026deg;C to construct AuCePt PHNs-LA. Finally, AuCePt PHNs-LA was stored at 4\u0026deg;C for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSOD-like activity of AuCePt PHNs measurement\u003c/h2\u003e \u003cp\u003eThe SOD-like activity of AuCePt PHNs was evaluated with a total superoxide dismutase assay kit (S0109, Beyotime Biotechnology, Shanghai, China). \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, generated by the reaction system of xanthine (X) and xanthine oxidase (XO), can reduce NBT to blue formazan, which has the strongest absorbance at 560 nm. Since SOD can scavenge \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, the SOD-like activity of AuCePt PHNs is inversely proportional to the production of blue formazan. Kinetic measurement: AuCePt PHNs (10 \u0026micro;L, 40 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or deionized water were mixed with the NBT working solution. The absorbance of the mixed solution at 560 nm was monitored for 30 min using a microplate reader [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. End-point detection: Different concentrations of Au PHNs, AuCe PHNs, AuPt PHNs and AuCePt PHNs were mixed with the NBT working solution. After incubation at 37 ℃ for 30 min, the absorbance at 560 nm of the mixture was detected using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCAT-like activity of AuCePt PHNs measurement\u003c/h2\u003e \u003cp\u003eFirst, the CAT-like activity of AuCePt PHNs was evaluated by monitoring the O\u003csub\u003e2\u003c/sub\u003e produced during H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition. Briefly, AuCePt PHNs (40 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (100 mM) were added to PBS (25 mL) at three different pH values (5.5, 6.5, and 7.4). Oxygen solubility (mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was measured within 300 s using a dissolved oxygen meter. In addition, the CAT-like activities of three similar nanomaterials (Au PHNs, AuCe PHNs and AuPt PHNs) and AuCePt PHNs were compared by measuring the dissolved oxygen yield under the same conditions.\u003c/p\u003e \u003cp\u003eIn the second method, the CAT-like activity of AuCePt PHNs was evaluated using a CAT assay kit (S0051, Beyotime Biotechnology, Shanghai, China). First, a standard curve was established by using different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0, 0.625, 1.25, 2.5, and 3.75 mM). Then, AuCePt PHNs and three similar nanomaterials (Au PHNs, AuCe PHNs and AuPt PHNs) were mixed with the precoordinated CAT reaction system. Finally, the absorbance of each sample at 520 nm was detected by a microplate reader after incubation in the dark at 25\u0026deg;C for 20 min. The residual H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was calculated as follows [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003eResidual H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e = (A\u003csub\u003e520\u003c/sub\u003e-b)/K\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetection of exogenous and endogenous ROS-scavenging activity\u003c/h2\u003e \u003cp\u003eThe intracellular ROS level was monitored by using an ROS assay kit (C1300-1, Applygen Technologies, Beijing, China). AML-12 cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells\u0026middot;well\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were seeded in 6-well plates for 24 h, and then these cells were treated with the following substances: 1) saline, 2) AuCePt PHNs (2 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 3) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (500 \u0026micro;M), and 4) AuCePt PHNs (2 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (500 \u0026micro;M). Moreover, after incubation with free fatty acids (FFAs) mixture (1 mM, at a 2:1 ratio of oleate/palmitate) for 20 h, AML-12 cells were treated with 5) saline and 6) AuCePt PHNs (2 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). After elution, these cells were incubated with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 \u0026micro;M) at 37\u0026deg;C for 30 min. Fluorescence images were recorded with a fluorescence microscope (Nikon ECLIPSE 80i, Japan), and the fluorescence intensity was measured with a Cary ECLIPSE fluorescence spectrophotometer (excitation: 502 nm, emission: 530 nm) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of DSF-loaded AuCePt PHNs\u003c/h2\u003e \u003cp\u003eDSF (100 mg) was dissolved in dimethyl sulfoxide (DMSO, 10 mL). Subsequently, AuCePt PHNs (80 mg) were added to the DSF solution and stirred for 24 h in a darkroom. The mixture was centrifuged (8000 rpm \u0026times; 20 min) and washed with DMSO 3 times to obtain AuCePt PHNs@DSF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH value on DSF release\u003c/h2\u003e \u003cp\u003eDSF concentrations were analyzed by HPLC, and standard curves were established as previously reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To explore the drug release efficiency and pH dependence, AuCePt PHNs@DSF powder was dissolved in different pH (5.0 and 7.4) media. The supernatant was collected by centrifugation at 0.5, 1, 3, 6, 12, 24, and 48 h, and the same volume of fresh release medium was supplemented immediately. The concentration of DSF in the supernatant was analyzed by HPLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDSF loading and entrapment rate of AuCePt PHNs\u003c/h2\u003e \u003cp\u003eThe drug loading and encapsulation rate of AuCePt PHNs were calculated by the following equation:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1719926544.png\"\u003e\u003cbr\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture, cytotoxicity test and construction of IR cell model\u003c/h2\u003e \u003cp\u003eAML-12, HepG-2, and HEK-293T cells were cultured in DMEM containing 10% fatal bovine serun (FBS) and 1% penicillin‒streptomycin at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. For the cytotoxicity test, AML-12 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells\u0026middot;well\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were plated in 96-well plates and treated with AuCePt PHNs (0, 10, 20, 50, 100 and 500 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) or DSF (0, 2, 4, 6, 8 and 10 \u0026micro;M) for 3, 6, 12, 24, 48 and 72 h. Cell viability was measured using CCK-8 (Beyotime, Shanghai, China) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To establish a lipid metabolism disorder or IR cell model, cells were incubated with FFAs mixture at 37\u0026deg;C for 20 h. For insulin signaling studies, cells were stimulated by insulin for 30 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFor detection of apoptosis, AML-12 cells were labeled with annexin V and PI (Eliret Biotechnology Co., Ltd), as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Cells were acquired using multicolor flow cytometry (Beckman counter, DxFlex), and the fluorescence-activated cell sorting (FACS) data were analyzed using FlowJo (V10) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGlucose uptake assay\u003c/h2\u003e \u003cp\u003eAML-12 IR cells treated with FFAs mixture were cultured in DMEM containing 1) control, 2) IR cells, 3) DSF (8 \u0026micro;M), 4) AuCePt PHNs-LA (2 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 5) DSF\u0026thinsp;+\u0026thinsp;AuCePt PHNs-LA (2 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and 6) AuCePt PHNs-LA@DSF (2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 12 h. Cells were then collected and cultured in glucose-free DMEM for 30 min, and 2-NDBG (200 \u0026micro;M) was added for another 20 min. After washing three times, fluorescent images of intracellular 2-NBDG were obtained by a fluorescence microscope (Nikon ECLIPSE 80i), and the fluorescence intensity was measured at 540 nm by a fluorescence spectrophotometer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blots\u003c/h2\u003e \u003cp\u003eWestern blots were performed as previously reported [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Primary antibodies included anti-AKT/anti-phospho-AKT (9272S/4060S, Cell Signaling Technology), anti-IRS-1/anti-phospho-IRS-1 (2382S, Cell Signaling Technology), anti-PEPCK (sc-130,388, Santa Cruz), anti-FOXO-1 (2880S, Cell Signaling Technology), and anti-β-actin (17AV0410, ZSGB-BIO Inc.). Secondary antibodies (Multisciences Biotech, Hangzhou, China) were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence imaging\u003c/h2\u003e \u003cp\u003eTo verify the targeting of AuCePt PHNs-LA in vivo, fluorescence images were obtained. Briefly, AuCePt PHNs-LA (1 mL, 1 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was mixed with Cy7.5 (0.3 mL, 0.5 mg\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to form AuCePt [email protected]. The mixture was collected by centrifugation, washed twice with PBS, and redissolved in PBS solution (200 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). WT mice were given AuCePt [email protected] (200 \u0026micro;L) via tail vein injection. Fluorescence imaging was performed by a multispectral fluorescence system (visque in vivo smart, viewers, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and treatments\u003c/h2\u003e \u003cp\u003eTo establish a diet-induced IR animal model, eight-week-old male C57BL/6J (WT) mice were fed a high-fat diet (HFD, 60 kcal% fat, D12492, Research Diets, New Brunswick, NJ) for 12 weeks. Subsequently, mice were randomly divided into four groups, and the following substances were given once every other day by tail vein for 16 days: 1) HFD (saline, 0.2 mL), 2) DSF (3 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.2 mL), 3) AuCePt PHNs-LA (3 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.2 mL), and 4) AuCePt PHNs-LA@DSF (3 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.2 mL). For the intervention study in an animal model of diabetes, 8-week-old ob/ob mice (Jicui Yaokang Biotechnology, Jiangsu, China) were fed a standard diet (SD) for 2 weeks. Subsequently, mice were divided into two groups. and the following substances were given once every other day for 16 days: 1) SD (saline, 0.2 mL), 2) AuCePt PHNs-LA@DSF (3 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.2 mL). Body weight was continuously monitored for 16 days. After the experiment, the mice were killed, and the tissues were stored at -160\u0026deg;C for subsequent experiments. The animal experiments were approved by the animal research committee of Chongqing Medical University and in accordance with the NIH Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIndirect calorimetry and biochemical parameter analyses\u003c/h2\u003e \u003cp\u003eIndirect calorimetry was performed in a Comprehensive Lab Animal Monitoring System (MM-100; CWE, Ardmore, PA, USA). Body weight, heat and rectal temperature were measured at the indicated duration. The RER was calculated by 24-h VO\u003csub\u003e2\u003c/sub\u003e and 24-h VCO\u003csub\u003e2\u003c/sub\u003e as described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Blood creatinine (Cre), urea nitrogen (BUN), creatine kinase (CK), hemoglobin (HGB), serum ALT and AST were determined by an automated biochemistry analyzer (ADVIA Chemistry XPT, Siemens Healthcare DiagnosticsInc, USA), as previously reported [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGTT and ITT tests\u003c/h2\u003e \u003cp\u003eFor glucose tolerance test (GTT), mice were fasted overnight for 12 h and intraperitoneally injected with 20% glucose solution (7.5 \u0026micro;L\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Then, the concentration of glucose solution was adjusted so that the injection volume of each mouse was equal. For insulin tolerance test (ITT), mice were intraperitoneally injected with insulin (0.75 U\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Novolin) after fasting for 4 h, and blood glucose was measured at the designated time points as previously reported [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistological examination\u003c/h2\u003e \u003cp\u003eFormalin-fixed livers from mice were processed, and paraffin sections (5 \u0026micro;m) were stained with H\u0026amp;E. Frozen liver sections were stained with 0.15% Oil Red O according to standard procedures as previously reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean or standard deviation, and statistical analyses were performed using SPSS 20.0 software (Chicago, IL, USA). Statistical significance was determined by one-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test or unpaired Student\u0026rsquo;s t test. When p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, statistically significant differences were considered.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of AuCePt PHNs\u003c/h2\u003e \u003cp\u003eAuCePt PHNs were synthesized by sacrificial galvanic replacement of Co nanoparticles in the presence of HAuCl\u003csub\u003e4\u003c/sub\u003e, K\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e6\u003c/sub\u003e and Ce(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e. Transmission electron microscopy (TEM) showed that AuCePt PHNs were spherical particles with a porous surface, hollow structure and average diameter of ~\u0026thinsp;60 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which is conducive to its passing through the hepatic sinusoid with a pore diameter of 50\u0026ndash;180 nm of endothelial cells to reach hepatocytes or hepatic stellate cells [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Dynamic light scattering (DLS) experiments confirmed that the hydrodynamic size of AuCePt PHNs was ~\u0026thinsp;66 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The hydrodynamic diameter measured by DLS was slightly larger than that measured by TEM, which can be assigned to the fact that the size measured by DLS consisted of the diameters of both AuCePt PHNs and the hydration layer. The elemental mapping and energy dispersive X-ray spectroscopy (EDS) results showed that AuCePt PHNs were mainly composed of Au, Ce and Pt elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). X-ray photoelectron spectroscopy (XPS) spectra of AuCePt PHNs also showed the characteristic peaks of Au, Ce and Pt elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, the high-resolution XPS spectra fitted the characteristic peaks of each element in AuCePt PHNs (Figure S2), which showed Au containing 92.62% Au\u003csup\u003e0\u003c/sup\u003e, Ce containing 28.69% Ce\u003csup\u003e3+\u003c/sup\u003e and 71.31% Ce\u003csup\u003e4+\u003c/sup\u003e, and Pt containing 85.35% Pt\u003csup\u003e0\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHigh-resolution TEM was used to characterize the lattice spacing of the prepared materials. As shown in Figure S3A, the lattice spacing of AuCePt PHNs includes d [111]\u0026thinsp;=\u0026thinsp;0.23 nm and d [200]\u0026thinsp;=\u0026thinsp;0.20 nm, indicating the high crystallinity of AuCePt PHNs. The X-ray diffraction pattern (XRD) revealed the crystal planes of AuCePt PHNs, including the (111), (200), (220), (311) and (222) crystal planes, corresponding to 2θ values of 38.3\u0026deg;, 44.7\u0026deg;, 64.9\u0026deg;, 77.7\u0026deg; and 81.7\u0026deg;, respectively (Figure S3B). Compared with the standard cards (Au//PDF # 04-0784, Ce//PDF # 31\u0026ndash;0325 and Pt//PDF # 04-0802) and XRD pattern of Au PHNs, the diffraction peaks of AuCePt PHNs were slightly shifted, revealing that the Au, Ce and Pt species formed a homogeneous and single-phase alloy structure [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pore structure of AuCePt PHNs was analyzed by N\u003csub\u003e2\u003c/sub\u003e adsorption experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The results showed that AuCePt PHNs had two main pore sizes of 3.5 nm and 6.1 nm. Moreover, the high-resolution TEM image also clearly showed a pore with a diameter of approximately 3.2 nm (Figure S3A, red arrow), which is mutually confirmed with the results of the N\u003csub\u003e2\u003c/sub\u003e adsorption experiment.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ePreparation and characterization of AuCePt PHNs-LA\u003c/h2\u003e \u003cp\u003eBecause LA can specifically bind asialoglycoprotein receptor (ASGPR) on the surface of the hepatocyte membrane [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], AuCePt PHNs were modified with LA to target hepatocytes. L-cys with sulfhydryl and amino groups was used to link AuCePt PHNs and LA \u003cem\u003evia\u003c/em\u003e Au-S and amide bonds. To analyze this process, the morphological changes of AuCePt PHNs were first characterized by TEM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, after being modified by L-cys, the surface of AuCePt PHNs clearly showed a transparent film. In addition, the transparent film became thicker with further connection of LA. The DLS results also showed that the diameter of AuCePt PHNs increased with the stepwise modification of L-cys and LA. Zeta-potential results (Figure S4) showed that the surface potential of AuCePt PHNs decreases from ~ -15 to ~ -20 mV and then to ~ -33 mV after the modification of L-cys and LA. These results confirmed the successful grafting of L-cys and LA. A previous study proved that the electrostatic repulsion of nanoparticles with zeta potentials greater than 20 mV or less than \u0026minus;\u0026thinsp;20 mV was sufficient to maintain the stability of nanoparticles in solvents [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Thus, AuCePt PHNs-LA with an ~ -33 mV surface charge may have good stability and dispersion in the circulating system. As expected, the diameter of AuCePt PHNs remained stable after being stored in PBS or serum for 1 month, indicating good stability and dispersion of AuCePt PHNs under physiological conditions (Figure S5).\u003c/p\u003e \u003cp\u003eNext, \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance (\u003csup\u003e13\u003c/sup\u003eC-NMR) was used to further confirm the successful modification of LA on AuCePt PHNs. Because AuCePt PHNs did not contain elemental carbon, there was no signal peak in the scanning spectrum of AuCePt PHNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). After AuCePt PHNs were linked with L-cys and LA, signal peaks appeared at 24.7899, 25.2058, 42.6966\u0026thinsp;~\u0026thinsp;103.4555, 172.3389 and 176.5229 ppm. According to the report [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and the \u003csup\u003e13\u003c/sup\u003eC-NMR scanning spectrum of L-cys and LA, the peaks at 61.0013\u0026ndash;103.4555 ppm belong to LA, and the peaks at 24.7899, 55.895, 172.3389 ppm belong to L-cys, which confirmed that we successfully obtained the novel AuCePt PHNs-LA compounds.\u003c/p\u003e \u003cp\u003eIn addition, the changes in the functional groups of AuCePt PHNs, L-cys, and LA before and after connection were analyzed by Fourier transform infrared spectroscopy (FTIR). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, L-cys had S-H stretching tensile vibration (blue frame) at 2550\u0026ndash;2750 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, but the peak disappeared after L-cys combined with AuCePt PHNs. This result indicated that the sulfhydryl of L-cys formed a more stable Au-S bond with AuCePt PHNs [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and AuCePt PHNsPHN-L-cys was prepared. In addition, the vibration characteristic peak of C\u0026thinsp;=\u0026thinsp;O moved from 1741.03 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1634.29 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (red frame), which indicated that the -COOH of LA had successfully condensed with -NH\u003csub\u003e2\u003c/sub\u003e to form an amide bond [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and proved that LA was modified on the surface of AuCePt PHNs-L-cys. Therefore, L-cys connects AuCePt PHNs and LA through gold sulfur bonds and amide bonds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-like activity of AuCePt PHNs\u003c/h2\u003e \u003cp\u003eSOD catalyzes \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to generate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e, which is the initial step of the scavenging ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To evaluate the SOD-like activity of AuCePt PHNs, their performance in eliminating \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e produced by xanthine (X) and xanthine oxidase (XO) reaction systems was monitored. The kinetic curve of absorbance at 560 nm in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows that the absorbance rises very slowly in the presence of AuCePt PHNs. This is because AuCePt PHNs can efficiently eliminate \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and thus inhibit the reduction of NBT to blue methadone, indicating that AuCePt PHNs have excellent SOD-like activity. In addition, by comparing the SOD-like activity of AuCePt PHNs with three other similar nanomaterials (AuCe PHNs, AuPt PHNs and Au PHNs), AuCePt PHNs have the highest SOD-like activity at any concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is not only the product of \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e but also another important ROS. It can be catalyzed by CAT to generate O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO, which is the second key step in the ROS removal process (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Thus, the CAT-like activity of AuCePt PHNs was evaluated by monitoring the generation of O\u003csub\u003e2\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, AuCePt PHNs could efficiently catalyze H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate O\u003csub\u003e2\u003c/sub\u003e, and their CAT-like activity was the highest under neutral pH conditions. In addition, the O\u003csub\u003e2\u003c/sub\u003e generation rate increases with increasing AuCePt PHNs concentration. The oxygen production efficiency proved that the CAT-like activity of AuCePt PHNs was the highest among all nanomaterials (Figure S6A and B). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF shows that the residual amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the AuCePt PHNs group was significantly lower than that in the Au PHNs, AuPt PHNs or AuCe PHNs groups, indicating that the AuCePt PHNs have the strongest enzyme activity for catalyzing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition.\u003c/p\u003e \u003cp\u003eThese results indicated that AuCePt PHNs present both SOD and CAT-like activities and can catalyze cascade enzyme reactions to eliminate ROS (\u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e --- H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e --- O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO). The outstanding enzymatic activity of AuCePt PHNs can be attributed to the following aspects: (1) \u0026bull;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e readily captures protons from water, forming HO\u003csub\u003e2\u003c/sub\u003e\u0026bull; and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e. The adsorption of HO\u003csub\u003e2\u003c/sub\u003e\u0026bull; on the Au(111) and Pt(111) planes is a highly exothermic process, and the activation energy barriers of the Au(111) and Pt(111) planes are extremely low. Once HO\u003csub\u003e2\u003c/sub\u003e\u0026bull; is adsorbed on the surface, Au and Pt exert SOD-like activity, converting HO\u003csub\u003e2\u003c/sub\u003e\u0026bull; into O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 [47]\u003c/sub\u003e. (2) Under alkaline conditions, the Au(111) and Pt(111) planes can pre-adsorb OH, which serves as the active site to initiate the acid-like decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and can promote the conversion of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2 [18]\u003c/sub\u003e. (3) Cerium nanomaterials have a redox pair that can cycle between the +\u0026thinsp;3 and +\u0026thinsp;4 states of oxygen vacancy sites, providing excellent SOD/CAT-like activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(C and F) confirm our hypothesis that the combination of the three components would increase the enzyme-like activity of the AuCePt PHNs compared to that of single-component or two-component nanozymes.\u003c/p\u003e \u003cp\u003eTo investigate the ability of AuCePt PHNs to scavenge ROS at the cellular level, DCFH-DA was used as a fluorescent probe. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and Figure S7 (A and B), treatment with AuCePt PHNs did not lead to significant changes in ROS levels in normal cells. After H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, the fluorescence intensity was significantly increased, indicating that intracellular ROS levels were increased. However, the fluorescence was significantly reduced with the addition of AuCePt PHNs. These results suggested that AuCePt PHNs could scavenge exogenous ROS. To further explore the activity of AuCePt PHNs in scavenging endogenous ROS. Alpha mouse liver (AML-12) cells were treated with FFAs mixture to fabricate the IR model, which resulted in a significant increase in ROS levels (compared with normal cells). When AuCePt PHNs were added, the ROS level was significantly reduced, indicating that AuCePt PHNs effectively eliminated endogenous ROS.\u003c/p\u003e \u003cp\u003eAccording to literature reports [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], since SOD and CAT are distributed in different organelles (Figure S8), the production and degradation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under natural conditions need to be transported between organelles. However, AuCePt PHNs have excellent SOD- and CAT-like activities simultaneously, which can catalyze cascade reactions, thus saving the transport process of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e between organelles. It is thus clear that the cascade response activity of AuCePt PHNs to ROS may be very helpful to improve the scavenging efficiency of ROS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLoading capacity and release efficiency of AuCePt PHNs for DSF\u003c/h2\u003e \u003cp\u003eIn the above experiments, it has been confirmed that AuCePt PHNs have a porous and hollow physical structure. The literature has reported that this special physical structure endows nanoparticles with a strong load capacity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. According to the fitting linear equations of DSF detected by high-performance liquid chromatography (HPLC) (Figure S9) and the calculation equation in section 2.8, the loading rate of AuCePt PHNs for DSF was 37.3% \u0026plusmn; 2.9, and the entrapment rate was 47.8% \u0026plusmn; 5.8.\u003c/p\u003e \u003cp\u003eNext, to analyze the release efficiency of AuCePt PHNs for DSF at different pH values, the cumulative release rate of DSF in 0\u0026ndash;48 h was measured. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows that the release of DSF from AuCePt PHNs is mainly concentrated within 24 h and has a significant pH dependence. When the pH of the dissolution medium was 7.4, the release efficiency in 48 h was only 36.98%, while when the pH was 5.5, the release efficiency reached approximately 74.76%. The pH dependence can reduce the nonspecific release of AuCePt PHNs to drugs in peripheral blood (pH\u0026thinsp;=\u0026thinsp;7.35\u0026ndash;7.45), while some organelles with lower pH (such as lysosomes) can promote the release of DSF from AuCePt PHNs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity and intracellular distribution of AuCePt PHNs\u003c/h2\u003e \u003cp\u003eTo investigate the cytotoxicity of AuCePt PHNs and DSF, we first used Cell Counting Kit-8 (CCK-8) in AML-12 cells (Figure S10A and B). The results showed no significant cytotoxicity when the concentrations of AuCePt PHNs and DSF were less than 100 \u0026micro;g/mL and 8 \u0026micro;M, respectively. In addition, flow cytometric analysis further confirmed that there was no significant evidence of cell apoptosis in AML-12 cells after treatment with 100 \u0026micro;g/mL AuCePt PHNs or 8 \u0026micro;M DSF (Figure S11A and B). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB clearly shows that AuCePt PHNs-LA was engulfed by cells through endocytosis and was encapsulated in endosomes. Importantly, the membrane and nuclear structures of the AuCePt PHNs-LA-treated AML-12 cells were clear and complete, which also proved that AuCePt PHNs-LA has good histocompatibility. In addition, we found that after AuCePt PHNs-LA was endocytosed, these endocytic particles were uniformly dispersed, which effectively avoided aggregation-induced cytotoxicity.\u003c/p\u003e \u003cp\u003eTo study the intracellular distribution of AuCePt PHNs-LA, AML-12 cells were treated with LysoTracker (red) and AuCePt PHNs-LA loaded with coumarin 6 (AuCePt PHNs-LA@cou6, green), and fluorescence colocalization analysis was performed at different time points. The results showed that the longer the treatment time of AuCePt PHNs-LA@cou6 was, the larger the yellow area (colocalization area) in cells, which peaked at 6 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). This phenomenon indicated that the amount of AuCePt PHNs-LA entering the lysosome was the largest after incubation for 6 h. However, when the cells were incubated for 12 h, the green fluorescence and red fluorescence were obviously separated, indicating that some AuCePt PHNs-LA had escaped from the lysosome. The results of intracellular distribution and fluorescence colocalization indicated that AuCePt PHNs-LA could enter the lysosome so that DSF was effectively released in the acidic environment and could escape from the lysosome to play a therapeutic role in the cytoplasm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEffects of AuCePt PHNs-LA@DSF on intracellular IR\u003c/h2\u003e \u003cp\u003eThe researches of Prof. Mailloux and Prof. Sun proved that DSF can clear ROS and reduce OS [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Furthermore, the above experiments confirmed that AuCePt PHNs-LA can reduce the endogenous and exogenous ROS. Therefore, we speculate that AuCePt PHNs-LA and DSF can jointly improve IR by reducing OS. To explore the effect of AuCePt PHNs-LA@DSF on cell-level IR, we conducted a 2-NBDG uptake experiment in AML-12 IR cells. Cell fluorescence imaging showed that the fluorescence intensity of the four treatment groups (DSF, AuCePt PHNs-LA, and AuCePt PHNs-LA@DSF) was higher than that of control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This finding suggested that they can improve glucose uptake in IR cells. Among them, AuCePt PHNs-LA@DSF group had the highest glucose uptake rate. Similar results were obtained by flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, the glycogen staining results showed that the glycogen content in IR cells treated with AuCePt PHNs-LA and DSF was significantly increased, while the cell glycogen content was the highest after AuCePt PHNs-LA@DSF treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Therefore, the AuCePt PHNs-LA@DSF can efficiently promote glucose uptake and glycogen synthesis in IR cells, thereby improving glucose metabolism.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of AuCePt PHNs-LA@DSF on gluconeogenesis and insulin signaling molecules in IR hepatocytes\u003c/h2\u003e \u003cp\u003eIt has been well documented that OS promote the occurrence and development of IR through insulin signaling pathway and gluconeogenesis signaling pathway [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, we next explored the influence of AuCePt PHNs-LA@DSF on the phosphorylation and expression of key molecules in these two classic pathways, including IRS-1, AKT (a serine/threonine kinase and a versatile node in insulin signal transduction) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], FOXO-1 (an important substrate molecule downstream of IRS-1/AKT, mediating gluconeogenesis) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and PEPCK (a rate-limiting enzyme in the gluconeogenesis pathway) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Firstly, we investigated the effects of different concentrations of Au and DSF on AKT phosphorylation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H, AuCePt PHNs-LA or DSF improved AKT phosphorylation levels in IR AML-12 cells in a dose-dependent manner. Secondly, by comparing the effects of different nanomaterials on the AKT phosphorylation level, it could be seen that AuCePt PHNs had the highest regulation efficiency (Figure S12). Significantly, treatment with DSF, AuCePt PHNs-LA, AuCePt PHNs-LA@DSF resulted in increased IRS-1 and AKT phosphorylation levels, and decreased FOXO1 and PEPCK expression in IR AML-12 cells. Especially, the effect of AuCePt PHNs-LA@DSF is the best (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u0026thinsp;~\u0026thinsp;M). These results indicated that AuCePt PHNs-LA@DSF can improve IR by regulating the insulin signaling pathway and the gluconeogenesis signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eTargeting efficiency, biodistribution and toxicity of AuCePt PHNs-LA in vivo\u003c/h2\u003e \u003cp\u003eTo investigate whether the LA-modified AuCePt PHNs can promote the phagocytosis of hepatocytes, we used doxorubicin (DOX) to label AuCePt PHNs and AuCePt PHNs-LA to construct fluorescent probes. AML-12 or HEK293T cells were treated with AuCePt PHNs or AuCePt PHNs-LA as indicated in the Methods. We found that the fluorescence signal in AML-12 cells treated with AuCePt PHNs-LA@DOX was significantly higher than that in AuCePt PHNs@DOX-treated AML-12 cells, indicating that LA modification improved the phagocytosis efficiency of AuCePt PHNs in AML-12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, in AuCePt PHNs-LA@DOX-treated cells, the fluorescence signal in HEK293T cells was significantly weaker than that in AML-12 cells, indicating that LA modification promoted the ASGPR-mediated endocytosis of AuCePt PHNs-LA, which is highly expressed on the surface of hepatocytes. Based on LA functional modification and the pH dependence of drug release, AuCePt PHNs-LA could efficiently target the liver to release DSF, thereby reducing the nonspecific distribution and toxicity of nanomaterials and drugs.\u003c/p\u003e \u003cp\u003eTo understand the biodistribution of AuCePt PHNs-LA in vivo, WT mice were injected with Cy7.5-labeled AuCePt PHNs or AuCePt PHNs-LA via the tail vein. In vivo fluorescence imaging showed that AuCePt [email protected] accumulated more in the liver than did AuCePt [email protected] after 3h of injection, suggesting that AuCePt PHNs-LA were more targeted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Consistently, organ fluorescence imaging also confirmed this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Figure S13). We continued to explore the distribution of AuCePt PHNs-LA in organs at different time points. The fluorescence of the liver decreased gradually over time. Importantly, the liver still retained a strong fluorescence signal after 48 h, which indicated that AuCePt PHNs-LA can stay in the liver for a long time to exert its enzyme-like activity. The fluorescence intensity in the kidney increased, suggesting that the material may be excreted through the kidney.\u003c/p\u003e \u003cp\u003eNext, to verify the toxicity of DSF and AuCePt PHNs-LA in vivo, we analyzed routine blood and biochemical indicators in mice. Compared with HFD group, DSF, AuCePt PHNs-LA, and AuCePt PHNs-LA@DSF treatment did not significantly affect various indicators in the mice (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In addition, there was no significant change in H\u0026amp;E staining of various tissues or organs (Figure S14). These results indicated that AuCePt PHNs-LA@DSF did not cause cardiac or renal dysfunction or systemic functional disorders in mice. Therefore, AuCePt PHNs-LA@DSF has good biosecurity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of AuCePt PHNs-LA@DSF on energy expenditure in vivo\u003c/h3\u003e\n\u003cp\u003eTo establish HFD-induced obese animal models, WT mice were fed a HFD for 12 weeks and treated with DSF, AuCePt PHNs-LA, or AuCePt PHNs-LA@DSF for 16 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Compared with the HFD group, the mice in the treatment groups were significantly smaller body weight and in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). In particular, the weight loss was most obvious in the AuCePt PHNs-LA@DSF-treated group. In addition, the morphological, H\u0026amp;E staining and oil-red O staining images of the liver showed that lipid deposition was significantly reduced in the treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and Figure S15\u0026thinsp;~\u0026thinsp;16). Similarly, among several treatments, the AuCePt PHNs-LA@DSF treatment was the most effective at reducing lipid deposition.\u003c/p\u003e \u003cp\u003eOne of the important reasons for obesity is an imbalance in energy metabolism, that is, an increase in energy storage and a decrease in energy consumption. Therefore, we measured the energy expenditure in each treatment group with metabolic cages. We found that oxygen consumption (VO\u003csub\u003e2\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) and carbon dioxide (VCO\u003csub\u003e2\u003c/sub\u003e) production (Figure S17A and B) increased significantly in the three treatment groups, indicating a higher metabolic rate. The respiratory exchange ratio (RER) also increased to varying degrees, especially in the AuCePt PHNsPHN-LA@DSF group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). A higher RER meant a higher utilization rate of carbohydrates, implying that IR was alleviated. Moreover, the whole-body energy expenditure and rectal temperature also increased significantly (Figure S17C and D). Overall, these data indicated that AuCePt PHNs-LA@DSF treatment significantly reduced body weight and liver lipid accumulation and increased overall energy consumption. Moreover, our data suggested that AuCePt PHNs-LA and DSF exhibit combined therapeutic effects. In addition, these results proved that the increase in energy consumption is the main cause of weight loss in the AuCePt PHNs-LA@DSF treatment group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eEffects of AuCePt PHNs-LA@DSF on glucose metabolism and IR in vivo\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of AuCePt PHNs-LA@DSF on insulin sensitivity in vivo, WT mice were fed a HFD for 12 weeks to construct an IR model and then treated with DSF, AuCePt PHNs-LA, DSF\u0026thinsp;+\u0026thinsp;AuCePt PHNs-LA or AuCePt PHNs-LA@DSF for 16 days via the tail vein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026thinsp;~\u0026thinsp;C, fasting blood glucose and insulin levels in the three treatment groups were markedly reduced. In particular, the fasting blood glucose and insulin levels of AuCePt PHNs-LA@DSF group mice were very close to those of the control group mice. Moreover, during the GTT and ITT experiments, the blood glucose level and the area under the glucose curve (AUC) at each time point in the three treatment groups significantly decreased, while those in the AuCePt PHNs-LA@DSF-treated group were the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026thinsp;~\u0026thinsp;G).\u003c/p\u003e \u003cp\u003eTo further explore the effect of AuCePt PHNs-LA@DSF on insulin sensitivity in the liver at the molecular level, we measured the level of phosphorylation or expression of insulin and gluconeogenesis signaling molecules extracted from liver tissues. Compared with the control group, the phosphorylation levels of IRS-1 and AKT were significantly increased in the liver, while the levels of PEPCK and FOXO1 proteins were significantly decreased, suggesting that insulin sensitivity was improved in the liver, especially in AuCePt PHNs-LA@DSF-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u0026thinsp;~\u0026thinsp;L).\u003c/p\u003e \u003cp\u003eChronic low-grade inflammation leads to liver steatosis, IR and obesity. Thus, the effect of AuCePt PHNs-LA@DSF on OS and inflammation in the liver was analyzed. The ROS-scavenging efficiency of each treatment group was measured using DCFH-DA as the fluorescent probe. We found that AuCePt PHNs-LA and DSF treatment significantly reduced ROS levels in the livers of HFD-fed C57BL/6J mice (Figure S18). The fluorescence intensity of the AuCePt PHNs-LA@DSF-treated group was the weakest, indicating that its antioxidant capacity was the strongest. In addition, we investigated the effects of AuCePt PHNs-LA@DSF on inflammatory factors and liver enzymes in vivo. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM\u0026thinsp;~\u0026thinsp;P, these three treatment groups all exhibited significant decreases in serum TNF-α and IL-6 levels and significant improvements in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. These changes were most obvious in the AuCePt PHNs-LA@DSF-treated group, suggesting that the combination of DSF and AuCePt PHNs-LA had the strongest anti-inflammatory effect and ameliorated liver injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy verification of AuCePt PHNs-LA@DSF on ob/ob mice\u003c/h2\u003e \u003cp\u003eOb/ob mice is a classic model of genetic obesity, accompanied by liver steatosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, to evaluate the effects of AuCePt PHNs-LA@DSF treatment on IR, obesity and fatty liver, ob/ob mice were treated with AuCePt PHNs-LA@DSF for 16 days via the tail vein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The results showed that AuCePt PHNs-LA@DSF treatment significantly reduced body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026thinsp;~\u0026thinsp;C, and Figure S19A), fasting blood glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), fasting insulin (Figure S19B), and liver lipid deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and Figure S19C). In addition, the glucose tolerance and insulin tolerance improved significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF\u0026thinsp;~\u0026thinsp;I). Moreover, ROS levels and inflammation in the livers of ob/ob mice were alleviated (Figure S19D). Western blot analysis revealed that compared with the SD-fed control group, the protein expression of PEPCK and FOXO1 in AuCePt PHNs-LA@DSF-treated ob/ob mice was significantly inhibited, while the phosphorylation of IRS-1 and AKT were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-N). These data indicated that AuCePt PHNs-LA@DSF treatment efficiently inhibited gluconeogenesis and improved insulin sensitivity in ob/ob mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we successfully synthesized a novel porous hollow cascade nanozymes, AuCePt PHNs, which were functionalized with LA and loaded with DSF to alleviate hepatic IR by reducing OS. AuCePt PHNs simultaneously had SOD/CAT-like activity and exhibited extremely strong loading and pH-dependent release capacity for DSF based on their porous and hollow structures, which effectively reduced the nonspecific release of DSF in the peripheral blood circulation. In vitro experiments proved that AuCePt PHNs-LA could improve glucose uptake and glycogen synthesis in AML-12 IR cells by scavenging endogenous and exogenous ROS, while this antioxidative effect was significantly enhanced after loading with DSF (AuCePt PHNs-LA@DSF). Studies in HFD-fed mice revealed that AuCePt PHNs-LA@DSF can significantly reduce blood glucose levels and improve liver lipid deposition, IR and obesity. Even in diabetic ob/ob mice, the above effects of AuCePt PHNs-LA@DSF were also obvious, suggesting that it has an excellent antidiabetic effect. In addition, compared with single administration, the better therapeutic efficacy of AuCePt PHNs-LA@DSF indicates that AuCePt PHNs-LA and DSF have combined effects in the field of antioxidant therapy. Therefore, this work illustrates that AuCePt PHNs-LA@DSF can improve insulin sensitivity in diabetic mice by modulating IRS-1/AKT and gluconeogenesis signaling pathways and provides a promising therapeutic strategy for OS-related obesity, fatty liver disease and diabetes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHW.S: Conceptualization, Methodology, Investigation, Formal analysis, Writing-Original Draft, Funding acquisition.YF.F: Resources, Investigation, Formal analysis, Writing-Original Draft. FF.L: Resources, Investigation, Writing - Review \u0026amp; Editing.WL.Z: Writing - Review \u0026amp; Editing. Y.Y: Resources.GY.Y: Conceptualization, Methodology, Supervision, Project administration, Writing - Review \u0026amp; Editing.ML.Y: Formal analysis, Writing - Review \u0026amp; Editing.L.L: Resources, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Natural Science Foundation for the Youth of China (No. 81902171), the National Natural Science Foundation of China (No. 82170816), and the Natural Science Foundation of Chongqing, China (cstc2020jcyj-msxmX0040).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang J, Ma J, Nie H, Zhang XJ, Zhang P, She ZG, Li H, Ji YX, Cai J. 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Diabetes. 2021;70:2492\u0026ndash;505.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Insulin resistance, Oxidative stress, Nanozymes, Disulfiram, Targeted drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-4580829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4580829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs the pathophysiological basis of type 2 diabetes mellitus (T2DM), insulin resistance (IR) is closely related to oxidative stress (OS) and inflammation, while nanozymes have a good therapeutic effect on inflammation and OS by scavenging reactive oxygen species (ROS). Hence, AuCePt porous hollow cascade nanozymes (AuCePt PHNs) are designed by integrating the dominant enzymatic activities of three metallic materials, which exhibit superior superoxide dismutase/catalase-like activities, and high drug loading capacity. In vitro experiments proved that AuCePt PHNs can ultra-efficiently scavenge endogenous and exogenous ROS. Moreover, AuCePt PHNs modified with lactobionic acid (LA) and loaded with disulfiram (DSF), named as AuCePt PHNs-LA@DSF, can significantly improve glucose uptake and glycogen synthesis in IR hepatocytes by regulating the insulin signaling pathways (IRS-1/AKT) and gluconeogenesis signaling pathways (FOXO-1/PEPCK). Intravenous administration of AuCePt PHNs-LA@DSF not only showed high liver targeting efficiency, but also reduced body weight and blood glucose and improved IR and lipid accumulation in high-fat diet-induced obese mice and diabetic ob/ob mice. This research elucidates the intrinsic activity of AuCePt PHNs for cascade scavenging of ROS, and reveals the potential effect of AuCePt PHNs-LA@DSF in T2DM treatment.\u003c/p\u003e","manuscriptTitle":"AuCePt porous hollow cascade nanozymes targeted delivery of disulfiram for alleviating hepatic insulin resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 13:26:49","doi":"10.21203/rs.3.rs-4580829/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-18T08:52:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T08:34:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-13T14:54:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-06T15:04:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100129882096886882070427379863436710050","date":"2024-07-06T14:36:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94576714041183583989178849422518016331","date":"2024-06-30T15:16:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298113655317544631983489246776880018477","date":"2024-06-27T10:59:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-25T08:32:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-15T15:51:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-15T15:50:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-06-14T09:00:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfed77ec-7213-4a88-b9fc-d611584c365e","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:02:30+00:00","versionOfRecord":{"articleIdentity":"rs-4580829","link":"https://doi.org/10.1186/s12951-024-02880-z","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-10-26 15:57:38","publishedOnDateReadable":"October 26th, 2024"},"versionCreatedAt":"2024-07-02 13:26:49","video":"","vorDoi":"10.1186/s12951-024-02880-z","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02880-z","workflowStages":[]},"version":"v1","identity":"rs-4580829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4580829","identity":"rs-4580829","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}