Metabolic dysfunction-associated steatohepatitis (MASH), also known as non-alcoholic steatohepatitis (NASH), is associated with the risk of developing hepatic cirrhosis and cancer [[1], [2], [3], [4], [5], [6], [7], [8]]. However, almost no drugs have been approved for chemotherapy. During the reviewing process of this paper, one of the new drugs, Rezdiffra®, was approved in March 2024. Although Rezdiffra® exhibits an antifibrosis effect in MASH patients, not all patients obtained the therapeutic outcomes (< 30%) [9]. In addition, the hepatological investigation and the long-term adverse effects have not been deeply assessed, yet. MASH can be triggered by various factors, including unhealthy lifestyle choices such as sedentary behavior and excessive caloric intake. The etiology of malnutrition-associated fatty liver, similar to MASH, involves severe protein and amino acid deficiency [[10], [11], [12]]. Therefore, expedited development MASH treatments become imperative. Although the adequate elucidation of the MASH mechanism remains unclear, the accumulation of substantial lipid deposits in the hepatic tissue attributable to MASH results in the excessive generation of reactive oxygen species (ROS), thereby inducing a state of pronounced oxidative stress [8,[13], [14], [15], [16]]. Excessive ROS generation stimulates the nuclear factor-kappa B (NF-κΒ) pathways, thereby initiating inflammation and satellite cell activation, ultimately leading the hepatic fibrosis [[17], [18], [19], [20], [21]]. Based on this mechanism, antioxidants such as N-acetylcysteine (NAC) have been proposed as candidate drugs, although none have been approved. This is attributed to the fact that both natural antioxidants and those developed to date, such as NAC, possess low molecular weight (LMW) and undergo rapid metabolism and excretion upon administration, necessitating frequent administration. Furthermore, the penetration of LMW antioxidants into normal cells disrupts the intracellular redox homeostasis, resulting in pronounced adverse effects. Consequently, conventional antioxidants exhibit a limited or nonexistent therapeutic window, rendering them ineffective as therapeutic agents. This is why it is very difficult to develop the medical drugs for MASH using small molecular antioxidants. Furthermore, the long-term adverse effect of Rezdiffra® has not been assessed well, and we must develop the safe MASH chemotherapy.

To address these issues with LMW antioxidants, we developed poly(L-cysteine) (PCys)-based amphiphilic block copolymers that spontaneously self-assemble into nanoparticles (NanoCys) in aqueous media and function as true antioxidants without side effects [22,23]. The amphiphilic block copolymers designed in this study contained poly(ethylene glycol) (PEG) as the hydrophilic segment and PCys with protected sulfanyl groups as the hydrophobic segment (PEG-block-PCys). PEG-block-PCys were engineered for spontaneous self-assembly into nanoparticles in aqueous environments. Upon ingestion, the polypeptide segments within the PCys segments undergo hydrolysis facilitated by endogenous enzymes, for example, such as peptidases in the small intestines and chymotrypsin excreted from pancreas, leading to the sustained release of Cys as an antioxidant.

In this study, hydrophobic tert-butyl thiol (StBu) and butyryl (Bu) groups were selected as protective moieties for the sulfanyl groups in the side chain of the PCys segment within PEG-block-PCys, which we separately reported [22,23]. Owing to their conjugation to polymers via disulfide and thioester groups, their distinct deprotection mechanisms respectively involve reduction and hydrolysis, and thioester groups have a high reactivity especially against the primary amino compounds to quickly produce the sulfanyl group. NanoCys, with a size in the tens of nanometers, mitigates cellular uptake, thereby reducing dysfunction of the intracellular redox balance. In fact, our NanoCys(SS), which was prepared using a dithiol protective block copolymer, suppressed severe adverse effects against murine colon carcinoma cells (colon-26) and human umbilical vein endothelial cells (HUVECs) [22]. We also confirmed that NanoCys(SS) administered intraperitoneally in mice, mitigates severe adverse effects in the gastrointestinal (GI) tract, in sharp contrast to the same dosage of NAC, which leads to pronounced bleeding in the GI tract [22]. Thus, the strategic design of self-assembly in amphiphilic PEG-block-PCys enabled a substantial reduction in the pronounced side effects associated with traditional LMW antioxidants. Simultaneously, the PCys chain was expected to be cleaved by endogenous enzymes, facilitating the release of cysteine. Indeed, we conducted assessments using cancer and sepsis models to evaluate oxidative stress-related diseases [22,23]. For instance, in a solid tumor xenograft mouse model, NanoCys(SS) administered intraperitoneally resulted in a significant suppression of tumor growth compared with the control and Cys-administration groups [22]. In another example, NanoCys(Bu), prepared using thioester-protective block copolymers, was orally administered to BALB/c mice via free drinking. Such a preparation remarkably extended the half-life by five to six hours after the induction of septic shock by lipopolysaccharide (LPS) compared to the control and Cys administration groups [23]. Thus, NanoCys is a promising candidate for the treatment of oxidative stress-related diseases, including cancer and sepsis.

Our next challenge is to engineer a suitable structure of the block copolymers, PEG-block-PCyses to improve their efficacy, suppressing their possible adverse effects. To develop a newly designed NanoCys system, we synthesized PEG-block-PCys block copolymers with the different protecting groups as well as the different chain lengths. Our focus was on utilizing MASH as a model, a condition in which conventional antioxidants exhibit limited therapeutic efficacy, as shown in Scheme 1. NanoCys(Bu), orally administered to MASH model mice, significantly suppressed lipid accumulation and oxidative damage in the liver compared to NanoCys(SS) as well as LMW Cys. Furthermore, shorter PCys chains exhibited better antioxidant effects than longer PCys chains, which could be explained by the in vitro liberation properties of Cys from NanoCyss facilitating proteases. Owing to its antioxidant function obtained by NanoCyses, NanoCyses ameliorated hepatic inflammation and had the promise of liver fibrosis suppression. According to our investigation, we elucidated that precision polymer design, including PCys chain length and side chain design, was the key to obtaining a significant outcome without the adverse effects of Cys in antioxidant chemotherapy.