Metabolic dysfunction-associated steatohepatitis treated by poly(ethylene glycol)-block-poly(cysteine) block copolymer-based self-assembling antioxidant nanoparticles.

Non-alcoholic steatohepatitis (NASH), now known as metabolic dysfunction-associated steatohepatitis (MASH), involves oxidative stress caused by the overproduction of reactive oxygen species (ROS). Small-molecule antioxidants have not been approved for antioxidant chemotherapy because of severe adverse effects that collapse redox homeostasis, even in healthy tissues. To overcome these disadvantages, we have been developing poly(ethylene glycol)-block-poly(cysteine) (PEG-block-PCys)-based self-assembling polymer nanoparticles (NanoCyses), releasing Cys after in vivo degradation by endogenous enzymes, to obtain antioxidant effects without adverse effects. However, a comprehensive investigation of the effects of polymer design on therapeutic outcomes has not yet been conducted to develop our NanoCys system for antioxidant chemotherapy. In this study, we synthesized different poly(L-cysteine) (PCys) chains whose sulfanyl groups were protected by tert-butyl thiol (StBu) and butyryl (Bu) groups to change the reactivity of the side chains, affording NanoCys(SS) and NanoCys(Bu), respectively. To elucidate the importance of the polymer design, these NanoCyses were orally administered to MASH model mice as a model of oxidative stress-related diseases. Consequently, the acyl-protective NanoCys(Bu) significantly suppressed hepatic lipid accumulation and oxidative stress compared to NanoCys(SS). Furthermore, we substantiated that shorter PCys were much better than longer PCys for therapeutic outcomes and the effects related to the liberation properties of Cys from these nanoparticles. Owing to its antioxidant functions, NanoCyses also significantly attenuated hepatic inflammation and fibrosis in the MASH mouse model.

An orally deliverable ornithine-based self-assembling polymer nanomedicine ameliorates hyperammonemia in acetaminophen-induced acute liver injury.

l-Ornithine (Orn) is a core amino acid responsible for ammonia detoxification in the body via the hepatic urea cycle. Clinical studies in Orn therapy have focused on interventions for hyperammonemia-associated diseases, such as hepatic encephalopathy (HE), a life-threatening neurological symptom affecting more than 80% of patients with liver cirrhosis. However, its low molecular weight (LMW) causes Orn to diffuse nonspecifically and be rapidly eliminated from the body after oral administration, resulting in unfavorable therapeutic efficacy. Hence, Orn is constantly supplied by intravenous infusion in many clinical settings; however, this treatment inevitably decreases patient compliance and limits its application in long-term management. To improve the performance of Orn, we designed self-assembling polyOrn-based nanoparticles for oral administration through ring-opening polymerization of Orn-N-carboxy anhydride initiated with amino-ended poly(ethylene glycol), followed by acylation of free amino groups in the main chain of the polyOrn segment. The obtained amphiphilic block copolymers, poly(ethylene glycol)-block-polyOrn(acyl) (PEG-block-POrn(acyl)), enabled the formation of stable nanoparticles (NanoOrn(acyl)) in aqueous media. We employed the isobutyryl (iBu) group for acyl derivatization in this study (NanoOrn(iBu)). In the healthy mice, daily oral administration of NanoOrn(iBu) for one week did not induce any abnormalities. In the mice exhibiting acetaminophen (APAP)-induced acute liver injury, oral pretreatment with NanoOrn(iBu) effectively reduced systemic ammonia and transaminases levels compared to the LMW Orn and untreated groups. The results suggest that the application of NanoOrn(iBu) is of significant clinical value with the feasibility of oral delivery and improvement in APAP-induced hepatic pathogenesis. STATEMENT OF SIGNIFICANCE: Liver injury is often accompanied by hyperammonemia, a life-threatening condition characterized by elevated blood ammonia levels. Current clinical treatments for reducing ammonia typically entail the invasive approach of intravenous infusion, involving the administration of l-ornithine (Orn) or a combination of Orn and L-aspartate. This method is employed due to the poor pharmacokinetics associated with these compounds. In our pursuit of enhancing therapy, we have developed an orally administrable nanomedicine based on Orn-based self-assembling nanoparticle (NanoOrn(iBu)), which provides sustained Orn supply to the injured liver. Oral administration of NanoOrn(iBu) to healthy mice did not cause any toxic effects. In a mouse model of acetaminophen-induced acute liver injury, oral administration of NanoOrn(iBu) surpassed Orn in reducing systemic ammonia levels and liver damage, thereby establishing NanoOrn(iBu) as a safe and effective therapeutic option.

Newly Designed Cysteine-Based Self-Assembling Prodrugs for Sepsis Treatment.

Reactive oxygen species (ROS) are essential signaling molecules that maintain intracellular redox balance; however, the overproduction of ROS often causes dysfunction in redox homeostasis and induces serious diseases. Antioxidants are crucial candidates for reducing overproduced ROS; however, most antioxidants are less effective than anticipated. Therefore, we designed new polymer-based antioxidants based on the natural amino acid, cysteine (Cys). Amphiphilic block copolymers, composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(cysteine) (PCys) segment, were synthesized. In the PCys segment, the free thiol groups in the side chain were protected by thioester moiety. The obtained block copolymers formed self-assembling nanoparticles (NanoCys(Bu)) in water, and the hydrodynamic diameter was 40-160 nm, as determined by dynamic light scattering (DLS) measurements. NanoCys(Bu) was stable from pH 2 to 8 under aqueous conditions, as confirmed by the hydrodynamic diameter of NanoCys(Bu). Finally, NanoCys(Bu) was applied to sepsis treatment to investigate the potential of NanoCys(Bu). NanoCys(Bu) was supplied to BALB/cA mice by free drinking for two days, and lipopolysaccharide (LPS) was intraperitoneally injected into the mice to prepare a sepsis shock model (LPS = 5 mg per kg body weight (BW)). Compared with the Cys and no-treatment groups, NanoCys(Bu) prolonged the half-life by five to six hours. NanoCys(Bu), designed in this study, shows promise as a candidate for enhancing antioxidative efficacy and mitigating the adverse effect of cysteine.

Design of cysteine-based self-assembling polymer drugs for anticancer chemotherapy.

Reactive oxygen species (ROS) play essential roles in the body, such as the production of energy in oxidative phosphorylation and signal transduction for homeostasis. Redox balance in biological systems gradually collapses due to various environmental factors, including aging and disease, and induces oxidative stress in the body. None of the natural or synthetic antioxidants have been approved clinically, owing to their adverse effects. Herein, we developed L-cysteine (Cys)-based polymer micelles as new self-assembling antioxidants to reduce the adverse effects of conventional antioxidants. Poly(ethylene glycol)-block-poly(L-cysteine) (PEG-block-PCys) was synthesized via anionic ring-opening polymerization. Because the free SH groups in the side chains of the PCys segment were protected by disulfide bonds, the obtained block copolymers were amphiphilic and formed polymer micelles (NanoCyss) of tens of nanometers in size in aqueous media. The stability of NanoCyss in the presence of bovine serum albumin (BSA) was increased by increasing the molecular weight (MW) of the PCys segments, which was analyzed using dynamic light scattering (DLS). The size and coagulation tendency of NanoCyss were also analyzed using DLS measurements by changing the pH and NaCl concentration. NanoCyss were confirmed to be less toxic both in vitro and in vivo than N-acetylcysteine (NAC) because of their size and biocompatible PEG surface layer. Intraperitoneal (i.p.) administration of NanoCyss to the tumor xenograft mouse model successfully suppressed tumor growth. Interestingly, this effect depended on the MW of the PCys segments.

Amphiphilic Poly[poly(ethylene glycol) methacrylate]s with OH Groups in the PEG Side Chains for Controlling Solution/Rheological Properties and toward Bioapplication.

Poly[poly(ethylene glycol) methacrylate]s with OH groups on the PEG side chains [poly(PEGOHMA)s] were synthesized using ruthenium-catalyzed living radical polymerization (Ru-LRP) to diversify the polymer design of PEGylated methacrylate-based copolymers. Poly(PEGOHMA)s could not be prepared using the approach previously reported for the synthesis of poly[poly(ethylene glycol) methyl ether methacrylate [poly(PEGMA)]; therefore, the polymerization was adapted for poly(PEGOHMA)s. As a result, both homopolymerization and random and block copolymerization of PEGOHMA with other hydrophobic monomers were successfully achieved, resulting in the preparation of amphiphilic random block and star polymers. The solution and bulk properties of PEGOHMA-based (co)polymers were markedly different from those of PEGMA-based (co)polymers. By reacting the OH groups with biotin, protein-poly(PEGOHMA) conjugates were successfully prepared; however, it was not possible to prepare protein-polymer conjugates using terminal biotinylated PEGMA-based copolymers, owing to the steric hindrance of the unreactive PEG side chains.

Unnatural Oligoaminosaccharides with N-1,2-Glycosidic Bonds Prepared by Cationic Ring-Opening Polymerization of 2-Oxazoline-Based Heterobicyclic Sugar Monomers.

Glycooligomers and glycopolymers (glycocompounds) play important roles in maintaining homeostasis in biological systems. Glycobiology is a burgeoning area in the elucidation of biological systems for which the molecular design of glycocompounds requires further diversification, including both natural and unnatural glycocompounds. Herein, we proposed a synthesis strategy based on the chain polymerization of deliberately designed sugar monomers. Unnatural oligoaminosaccharides comprising N-1,2-glycosidic bonds were synthesized without enzymes through the cationic ring-opening polymerization of 2-oxazoline-based heterobicyclic sugar monomers. To achieve this, a heterobicyclic monomer [Glc(MeOx)], comprising protected glucosamine (GlcN) and 2-methyl-2-oxazoline (MeOx) rings, was designed. This monomer was polymerized using a binary initiating system of tert-butyl iodide (t-BuI) and GaCl3 to afford oligo[Glc(MeOx)]. The resulting structure corresponded to the condensation product of GlcN with N-1,2-glycosidic bonds. After deprotection of oligo[Glc(MeOx)], the resulting oligoaminosaccharide had a secondary structure different to that of protected oligo[Glc(MeOx)]. Owing to the N-1,2-glycosidic bonds, the oligoaminosaccharide was not degraded by chitinase, which hydrolyzes the condensation product of GlcN with O-1,4-glycosidic bonds.

Star Polymer Gels with Fluorinated Microgels via Star-Star Coupling and Cross-Linking for Water Purification.

Two types of star polymer gels containing perfluorinated microgels were created as purification materials to separate polyfluorinated surfactants (e.g., perfluorooctanoic acid) from water. One macrogel is prepared by the radical coupling of fluorine and/or amine-functionalized microgel star polymers alone, while another is done by the radical cross-linking of the star polymers with poly(ethylene glycol) methyl ether methacrylate. Importantly, the reactive olefin remaining within the microgel cores was directly employed for both coupling and cross-linking reactions. Swelling properties of star polymer gels were effectively controlled by the latter cross-linking technique. Analyzed by small-angle X-ray scattering, a star-star coupling gel typically consists of a three-dimensional network where star polymers are sequentially connected with the microgels at the constant interval of about 20 nm. Owing to the fluorous and acid/base cooperative interaction, star polymer gels carrying fluorine/amine-functionalized microgels efficiently captured polyfluorinated surfactants in water and successfully afforded the removal from water via simple mixing and filtration.

LCST-Type Phase Separation of Poly[poly(ethylene glycol) methyl ether methacrylate]s in Hydrofluorocarbon.

Poly[poly(ethylene glycol) methyl ether methacrylate]s [poly(PEGMA)s] sharply and reversibly exhibited lower critical solution temperature (LCST)-type phase separation in 2H,3H-perfluoropentane (2HPFP). The cloud points decreased from 52 to 41 °C with increasing the PEG pendant length [-(CH2CH2O)mCH3: m = 4.5, 9, 19]. The cloud point was precisely controlled via the addition of perfluoroalkanes (e.g., perfluorooctane) to the 2HPFP solution: typically, it was inversely proportional to the amount of perfluorooctane in the mixture. The unique thermoresponsive solubility further afforded the temperature-mediated micellization of a block copolymer of PEG19MA and methyl methacrylate (MMA) in 2HPFP to uniquely give a PEG-core micelle with PMMA shell. Therefore, the LCST phase separation properties in the hydrofluorocarbon would open new vistas for thermoresponsive polymeric materials.

Fluorous microgel star polymers: selective recognition and separation of polyfluorinated surfactants and compounds in water.

Immiscible with either hydrophobic or hydrophilic solvents, polyfluorinated compounds (PFCs) are generally "fluorous", some of which have widely been employed as surfactants and water/oil repellents. Given the prevailing concern about the environmental pollution and the biocontamination by PFCs, their efficient removal and recycle from industrial wastewater and products are critically required. This paper demonstrates that fluorous-core star polymers consisting of a polyfluorinated microgel core and hydrophilic PEG-functionalized arms efficiently and selectively capture PFCs in water into the cores by fluorous interaction. For example, with over 10 000 fluorine atoms in the core and approximately 100 hydrophilic arms, the fluorous stars remove perfluorooctanoic acid (PFOA) and related PFCs in water from 10 ppm to as low as a parts per billion (ppb) level, or an over 98% removal. Dually functionalized microgel-core star polymers with perfluorinated alkanes and additional amino (or ammonium) groups cooperatively recognize PFOA or its ammonium salt and, in addition, release the guests upon external stimuli. The "smart" performance shows that the fluorous-core star polymers are promising PFC separation, recovery, and recycle materials for water purification toward sustainable society.

Arm-cleavable microgel star polymers: a versatile strategy for direct core analysis and functionalization.

Arm-cleavable microgel star polymers were developed, where the arm chains can readily be cleaved by acidolysis after the synthesis, allowing isolation of the core, direct analysis of its structure, and also the creation of functional nanometer-sized microgels. The key is to employ a macroinitiator (PEG-acetal-Cl) that carries an acetal linkage between a poly(ethylene glycol) arm chain and a chloride initiating site. From this, star polymers were synthesized via the linking reaction with a divinyl monomer and a ruthenium catalyst in living radical polymerization. The arms were subsequently cleaved by acidolysis of the acetal linker to give soluble microgels (cores free from arms). Full characterization revealed that the microgel cores are spherical, nano-sized (<20 nm), and of relatively low density. Amphiphilic, water-soluble, and thermosensitive arm-free microgels can be obtained by additionally employing functional methacrylate upon arm linking.

Sequence-regulated copolymers via tandem catalysis of living radical polymerization and in situ transesterification.

Sequence regulation of monomers is undoubtedly a challenging issue as an ultimate goal in polymer science. To efficiently produce sequence-controlled copolymers, we herein developed the versatile tandem catalysis, which concurrently and/or sequentially involved ruthenium-catalyzed living radical polymerization and in situ transesterification of methacrylates (monomers: RMA) with metal alkoxides (catalysts) and alcohols (ROH). Typically, gradient copolymers were directly obtained from the synchronization of the two reactions: the instantaneous monomer composition in feed gradually changed via the transesterification of R(1)MA into R(2)MA in the presence of R(2)OH during living polymerization to give R(1)MA/R(2)MA gradient copolymers. The gradient sequence of monomers along a chain was catalytically controlled by the reaction conditions such as temperature, concentration and/or species of catalysts, alcohols, and monomers. The sequence regulation of multimonomer units was also successfully achieved in one-pot by monomer-selective transesterification in concurrent tandem catalysis and iterative tandem catalysis, providing random-gradient copolymers and gradient-block counterparts, respectively. In contrast, sequential tandem catalysis via the variable initiation of either polymerization or in situ transesterification led to random or block copolymers. Due to the versatile adaptability of common and commercially available reagents (monomers, alcohols, catalysts), this tandem catalysis is one of the most efficient, convenient, and powerful tools to design tailor-made sequence-regulated copolymers.