Preventing acute liver injury via hepatocyte‐targeting nano‐antioxidants

Abstract Acute liver injury (ALI) is a severe liver disease that is characterized by sudden and massive hepatocyte necrosis and deterioration of liver functions. Oxidative stress is increasingly recognized as a key factor in the induction and progression of ALI. Scavenging excessive reactive oxygen species (ROS) with antioxidants has become a promising therapeutic option, but intrinsically hepatocyte‐targeting antioxidants with excellent bioavailability and biocompatibility are yet to be developed. Herein, self‐assembling nanoparticles (NPs) composed of amphiphilic polymers are introduced to encapsulate organic Selenium compound L‐Se‐methylselenocysteine (SeMC) and form SeMC NPs, which protect the viabilities and functions of cultured hepatocytes in drug‐ or chemical‐induced acute hepatotoxicity models via efficient ROS removal. After further functionalization with the hepatocyte‐targeting ligand glycyrrhetinic acid (GA), the resultant GA‐SeMC NPs exhibit enhanced hepatocyte uptake and liver accumulation. In mouse models of ALI induced by acetaminophen (APAP) or carbon tetrachloride (CCl4), treatment with GA‐SeMC NPs significantly decrease the levels of hepatic lipid peroxidation, tissue vacuolization and serum liver transaminases, while prominently increase that of endogenous antioxidant enzymes. Our study therefore presents a liver‐targeting drug delivery strategy for the prevention and treatment of hepatic diseases.

1][12] Therefore, scavenging excessive ROS has been considered as an accessible solution to improve the microenvironment of the injured liver and alleviate the pathological progression.Broad-spectrum antioxidants such as N-acetyl cysteine have exhibited preventive and therapeutic effects on ALI. 10,13wever, the fast excretion, unsatisfactory accumulation at the injury foci, and the resulting poor bioavailability largely limit their clinical applications. 10,14It is therefore highly desirable to develop more effective strategies focusing on liver-targeted ROS scavenging.6][17] Supplementation of Se has been shown to alleviate the tissue and organ damage induced by various toxic substances, including heavy metal ions, mycotoxins, antitumor drugs, etc. [18][19][20][21][22] Among various forms of Se compounds, the inorganic Se compounds are abundant and inexpensive, but exhibit low absorption and conversion rates as well as higher toxicity than organic Se compounds. 18,23,24L-Se-methylselenocysteine (SeMC), a new generation of organic Se compound, has shown protective effects on the organ damage caused by chemotherapy-induced systemic toxicity. 25However, the hepatoprotective effects of SeMCs against liver injury are not well explored.
[28][29][30][31][32] The resulting concept of 'nanomedicine' has enabled the development of new ROS scavenging strategies against ROS-related diseases using various functional nanomaterials. 14,33,34Nanoencapsulation of antioxidants by engineering amphiphilic micellar nanoparticles (NPs) has attracted great interest for the researchers due to their targeting properties and sustained-releasing features, which was regarded as a breakthrough in increasing the bioavailability and therapeutic efficacy of the antioxidants while reducing their potential side effects. 35,36spired by the advantages of amphiphilic micellar NP-based nanomedicine, nanoformulated SeMCs that were encapsulated by amphiphilic molecules and glycyrrhetinic acids (GA), namely GA-SeMC NPs, were generated in this study.We found that GA-SeMC NPs showed higher hepatocyte uptake and liver accumulation.In mouse models of ALI, these nano-antioxidants efficiently suppressed APAP-or CCl 4 -induced increases in the levels hepatic oxidative stress, histological lesions and serum transaminases, and promoted the expression of antioxidant enzymes, exhibiting superior hepatoprotective effects.

| Drug encapsulation efficiency (EE)
The amount of SeMC was measured using the absorbance of SeMC NPs at 570 nm according to the requirement of the ninhydrin chromogenesis method for the determination of amino acid content. 37e EE of SeMCs was calculated according to the following equations: is the weight of SeMC in NPs.

| In vitro cytotoxicity assay
Cells were cultured in 96-well plates at a density of 1 Â 10 4 cells per well and cultured for 12 h at 37 C.After incubation with SeMC NPs for 48 h, the cells were incubated with fresh media containing 10% CCK-8 solution for 30 min at 37 C.The viabilities of the cells were determined by measuring the absorbance at 450 nm with a microplate reader.

| In vitro cellular uptake
The hepatocyte-targeting ability of GA-SeMC NPs was evaluated with an in vitro cellular uptake assay.Rhodamine B was used as the fluo-  The capability of GA-SeMC NPs to ameliorate APAP-or CCl 4 -induced liver injury was compared to that of unmodified SeMC NPs and free SeMCs.
The mice were randomly divided into five groups (six mice per group).After the mice were anaesthetised, blood and liver samples were collected for further experiments.To determine liver injury in mice, ALT, AST, LDH activities were determined using the commercial assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), according to the manufacturer's instructions.

| Detection of SOD, GSH-Px and MDA activities
The liver homogenate was prepared, and the hepatic protein content was determined as described previously.The supernatant was used to determine the superoxide dismutase (SOD, A001-3) activity, glutathione peroxidase (GSH-Px, A005-1) activity and malondialdehyde (MDA, A003-1) content using commercial kits from Nanjing Jiancheng Bioengineering Institute, according to the manufacturer's instructions.

| Histopathology analysis
Fresh liver tissues from the same lobes were collected, trimmed to a thickness of about 2 mm, and then fixed in 10% buffered formaldehyde solution.Fixed tissues were cut into sections of 2 μm and stained using hematoxylin and eosin (H&E) followed by examination under a light microscope.

| Statistical Analysis
Statistical analyses were performed with the GraphPad Prism.All data were representative of ≥3 independent experiments and presented as mean ± SD or mean ± SEM.Student's t test was used to determine the statistical significance of the differences between two groups.ns means not significance, *p < 0.05, **p < 0.01, ***p < 0.001.

| Fabrication and characterization of SeMC NPs
GA-SeMC NPs were synthesized via a two-step procedure.

| Fabrication and characterization of hepatocyte-targeting GA-SeMC NPs
To endow the SeMC NPs with targeting ability for hepatocytes, we functionalized them with GA, a well-established liver targeting ligand, 40,41 and fabricated GA-SeMC NPs.The 1 H NMR spectra of GA, SeMC NPs, and GA-SeMC NPs were shown in Figure 4A.In comparison to unmodified SeMC NPs, the additional signal peak at $5.7 ppm that attributed to the protons of olefinic bond ( (C O) CH C ) in GA was detected in GA-SeMC NPs, demonstrating the conjugation of GA to the GA-SeMC NPs. 42,43To optimize the particle sizes and drug encapsulation efficiencies of the GA-SeMC NPs, different ratios of DSPE-PEG-GA to DSPE-PEG-NH 2 during the GA-SeMC NP preparation were tested.As oxidative stress is a critical factor in APAP-induced hepatotoxicity, the hepatic oxidative stress in liver tissue was evaluated by measuring the levels of malondialdehyde (MDA), an end product of lipid peroxidation that could reflect the levels of cellular oxidative stress. 46mpared with the untreated control group, APAP treatment significantly increased the hepatic MDA levels, suggesting that severe oxi- Normal liver cell line L-02 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai) and was maintained in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum (FBS, from Gibco) and antibiotics (streptomycin and penicillin, 100-U/ml each, Invitrogen).DSPE-PEG 2000 -OH, DSPE-PEG 2000 -NH 2 , PCL 1000 -PEG 2000 -NH 2 , PCL 2000 -PEG 5000 -NH 2 , PCL 5000 -PEI 2000 copolymers were purchased from Xi'an ruixi Biological Technology Co., Ltd.18β-Glycyrrhetinic acid was purchased from MedChemExpress (Shanghai).SeMC was provided by He'nan Xibaikang Health Industry Co., Ltd.Cell Counting Kit-8 (CCK-8) was purchased from Beyotime. 2 0 ,7 0 -dichlorofluorescin diacetate (DCFH-DA), Rhodamine B, acetaminophen (APAP) and carbon tetrachloride (CCl 4 ) were purchased from Sigma-Aldrich.

L-02 cells ( 2 Â
10 5 cells per well) were cultured in 24-well plates overnight.After incubation with SeMCs or SeMC NPs for 24 h, cells were washed with PBS and then incubate with APAP or CCl 4 for 12 h.After that, the cells were washed twice with PBS and incubated with RPMI 1640 containing 20 μM DCFH-DA for 40 min.Fluorescence imaging was performed on a Leica TCS SP8 confocal laser scanning microscope (Ex = 488 nm, Em = 520 nm).
rescent dye and observed by confocal laser scanning microscopy (CLSM; TCS SP8; Leica Microsystems).First, L-02 cells harvested in the exponential growth phase were seeded in 24-well plates at a density of 2 Â 10 5 cells/well and incubated overnight at 37 C. Subsequently, the cells were incubated with fresh RPMI 1640 containing RhB-labelled SeMC NPs or RhB-labelled GA-SeMC NPs for 1 or 1.5 h at 37 C.The cells were then washed three times with PBS and fixed with a 4% paraformaldehyde for 10 min at 25 C. Finally, the cells were observed by CLSM.

2. 8 |
Animal treatmentFemale BALB/c mice (6-8 weeks) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China).All mouse experiments were carried out according to protocols approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (A-2022-115).To explore the protective effect of GA-SeMC NPs in ameliorating APAP-or CCl 4 -induced liver injury, mice were intravenously injected with GA-SeMC NPs every other day for a total of three doses before APAP/CCl 4 treatment.The extent of liver injury was then determined by measuring serum levels of ALT and AST at 24 h after APAP (300 mg kg À1 ) or CCl 4 (10 mL kg À1 ) administration.

3 . 2 |
Figure S1A).The SeMC NPs and unloaded DSPE-PEG-NH 2 polymeric nanoparticles (PNPs) showed comparable positive surface potentials.After the loading of SeMCs, the sizes of NPs were slightly increased (Figure 2B,C).A period of five-week stability study showed no significant changes in particle size for the SeMC NPs, showing a decent stability (Figure S1B).To investigate the drug release characteristics of the SeMC NPs, the release profile of SeMCs from NPs at different pH values was evaluated using the dialysis method.As shown in Figure 2D, in both cytoplasmmimicking environment (pH 7.4) and the acidic endosome-mimicking environment (pH 5.5), SeMC NPs presented a fast releasing behaviour in the first 6 h, with the releasing levels of $36% at pH 7.4 and $41% at pH 5.5.Compared to the cytoplasmic environment, SeMC NPs had a slightly faster releasing rate under acidic condition, and the cumulative releasing levels reached approximately 82% in 24 h.Moreover, the biocompatibility of the synthesized SeMC NPs were evaluated by examining the cytotoxicity of these NPs in the cultured cells.Figure S2 exhibited that SeMC NPs had a negligible influence on the viabilities of L-02 normal liver cells at various SeMC concentrations (50-250 μg mL À1 ) within 48 h, indicating good biocompatibility of SeMC NPs.

F I G U R E 2
Figure S6A,B).In the meanwhile, the unloaded PNPs showed no effects on hepatocellular protection.Similarly, hepatocellular protective efficacy of SeMC NPs was also verified in CCl 4 -induced hepatocellular injury model.Pretreating L-02 cells with SeMC NPs for 24 h before CCl 4 treatment notably decreased the levels of secreted ALT and AST compared to CCl 4 treatment alone (Figure 3E and Figure S6C,D).Apart from ALT and AST, lactate dehydrogenase (LDH) is another index of hepatocyte function that is released during cell injury.Like the other two indexes, the uptake of SeMC NPs obviously reduced the levels of secreted LDH in the above-mentioned two experimental models of acute hepatocellular injury (Figure S7).Moreover, the protective effects of SeMC NPs towards hepatocytes under various stimulations were further verified by the CCK8 assays.As shown in the Figure S8, SeMC NPs significantly ameliorated the decrease of cell viabilities caused by APAP or CCl 4 , further verifying the protective effects of SeMC NPs against hepatocyte injury.

2 (
Figure S9A, the L-02 cells treated with RhB-labelled SeMC NPs showed much weaker intensities of the fluorescence comparing to the L-02 cells treated with RhB-labelled GA-SeMC NPs, reflecting the enhanced efficiency in the hepatocyte-targeting ability of GA-SeMC NPs.To verify the liver-targeting effects of GA-SeMC NPs in vivo, BALB/c mice were injected intravenously with RhB-labelled

3. 4 |
Figure S10, the serum levels ALT and AST in the mice were obviously increased 12 h after the injection of APAP, indicating successful establishment of APAP-induced ALI model.To explore the protective Figure 5F.Compared to the PBS-treated control group, severe hepatic lesions were observed in APAP-treated group, showing hepatic cell necrosis and vacuolization.Administration of SeMCs or SeMC NPs resulted in moderate improvement in liver histological lesions, while the mice injected with GA-SeMC NPs showed liver histological characteristics similar to those of normal healthy mice.To further verify the hepatoprotective effects of the GA-SeMC NPs, the mouse model of CCl 4 -induced ALI was used (FigureS12).As expected, the intraperitoneal injected CCl 4 led to significant elevation in the serum levels of ALT and AST, whereas pretreatment of GA-SeMC NPs markedly inhibited the elevation of these two indexes