Enhanced Stability of α-Mangostin-Rich Extract and Selective Cytotoxicity against Cancer Cells via Encapsulation in Antioxidant Nanoparticles (AME@NanoAOX).

α-Mangostin-rich extract (AME) shows promise as a functional ingredient for cancer chemotherapy. Here, we encapsulated AME in our originally designed antioxidant nanoparticles (NanoAOX) to increase its solubility and prevent oxidative degradation (AME@NanoAOX). In this study, two types of self-assembled polymers containing nitroxide radicals were engineered. These polymers were self-assembled into nanoscale particles in aqueous media, entrapping AME (abbreviated as AME@NanoAOX(B) and AME@NanoAOX(G)). These formulations considerably improved the stability of AME against oxidative degradation and exhibited different release profiles of α-mangostin under different pH conditions. Furthermore, AME-encapsulated nanoparticles exhibited potent cytotoxicity against various cancer cell lines, including human breast cancer (MCF-7), human lung cancer (A549), human colon cancer (Caco-2), human cervical cancer (HeLa), and human liver cancer (HepG2) cell lines, with minimal cytotoxicity in normal human mammary epithelial cells (hTERT-HME1), thus providing a high selectivity index (SI). These results indicated the promising feature of AME-encapsulated antioxidant nanoparticles (AME@NanoAOX) for cancer chemotherapy.

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.

Design of Antioxidant Nanoparticle, which Selectively Locates and Scavenges Reactive Oxygen Species in the Gastrointestinal Tract, Increasing The Running Time of Mice.

Excess reactive oxygen species (ROS) produced during strong or unfamiliar exercise cause exercise-induced gastrointestinal syndrome (EIGS), leading to poor health and decreased exercise performance. The application of conventional antioxidants can neither ameliorate EIGS nor improve exercise performance because of their rapid elimination and severe side effects on the mitochondria. Hence, a self-assembling nanoparticle-type antioxidant (RNPO ) that is selectively located in the gastrointestinal (GI) tract for an extended time after oral administration is developed. Interestingly, orally administered RNPO significantly enhances the running time until exhaustion in mice with increasing dosage, whereas conventional antioxidants (TEMPOL) tends to reduce the running time with increasing dosage. The running (control) and TEMPOL groups show severe damage in the GI tract and increased plasma lipopolysaccharide (LPS) levels after 80 min of running, resulting in fewer red blood cells (RBCs) and severe damage to the skeletal muscles and liver. However, the RNPO group is protected against GI tract damage and elevation of plasma LPS levels, similar to the nonrunning (sedentary) group, which prevents damage to the whole body, unlike in the control and TEMPOL groups. Based on these results, it is concluded that continuous scavenging of excessive intestinal ROS protects against gut damage and further improves exercise performance.

Efficacy of redox nanoparticles for improving survival of transplanted cells in a mouse model of ischemic stroke.

The success of cell transplantation therapy for ischemic stroke is hindered by the low cell survival rate in poststroke brain, due in part to high free radical production and ensuing oxidative stress. We have developed redox nanoparticles to eliminate reactive oxygen species. In this study, we tested the protective efficacy of these redox nanoparticles in cell culture and a mouse model of ischemic stroke. Induced human dental pulp stem cells were subjected to oxygen-glucose deprivation and reoxygenation to recapitulate ischemia and reperfusion in the penumbra surrounding a cerebral infarct. Cell viability using WST-8 assay, apoptosis using TUNEL, free radicals using MitoSOX, and inflammatory cytokines using ELISA kit were measured in the presence and absence of redox nanoparticles after oxygen-glucose deprivation and reoxygenation. The scavenging activity of redox nanoparticles against reactive oxygen species was detected by electron spin resonance. Moreover, induced cells were transplanted intracerebrally into to the distal middle cerebral artery occlusion model with and without redox nanoparticles, and the survival rate measured. Cell viability was enhanced, while apoptosis, free radical generation, and inflammatory cytokine expression levels were reduced in cultures with redox nanoparticles. Further, reduced redox nanoparticles were detected in the cytoplasm, indicating free radical scavenging. Addition of redox nanoparticles also improved the survival rate of transplanted cells after 6 weeks in vivo. These redox nanoparticles may increase the applicability and success of induced stem cell therapy for ischemic stroke patents by promoting long-term survival.

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.

Designing poly(gamma-aminobutyric acid)-based nanoparticles for the treatment of major depressive disorders.

Major depressive disorder (MDD) is a worldwide concern owing to its negative impact on the quality of life. Gamma-aminobutyric acid (GABA), an essential neurotransmitter in the brain, is important for regulating the enteric nervous system and gut-brain dual communication (gut-brain axis), thus providing gastrointestinal GABA and GABA-related pathways with possible targets for MDD treatment. However, the use of GABA for this disease remains limited due to its poor pharmacokinetic properties, including the low permeability through the blood-brain barrier, and the rapid clearance from the gastrointestinal tract. Since poly(amino acid)s are advantageous for improving the beneficial bioactivities of conventional amino acids, poly(gamma-aminobutyric acid) (poly(GABA)) is a potential candidate for MDD therapy. Nevertheless, the non-water-soluble and non-dispersible characteristics of poly(GABA) render difficulty in administering its conventional forms in vitro/in vivo, thereby hindering its therapeutic applications. Therefore, this study proposes a new design for poly(GABA) in nanoparticle form, which is composed of the amphiphilic diblock copolymers of poly(GABA) and poly(ethylene glycol), providing a suitable formulation for medication applications. Herein, we report on a new orally deliverable poly(GABA)-based nanoparticles (NanoGABA) in aqueous media and their efficacy on mouse depression models. NanoGABA treatment efficiently attenuated depression-like symptoms as evidenced by behavioral tests (forced swimming tests and tail suspension tests) and stress biomarkers (corticosterone). These findings suggest that the newly designed poly(GABA)-based nanoparticles are a promising candidate for the treatment of depression. STATEMENT OF SIGNIFICANCE: This research is the first to report the preparation of poly(GABA)-based nanoparticles in aqueous conditions with beneficial physical properties to open the gate for medical and pharmaceutical applications of poly (GABA). It is also a pioneer in using poly(GABA)-based materials for major depressive disorder therapeutics in vivo. Oral administration of NanoGABA attenuates depressive-like symptoms by targeting the enteric nervous system possibly through modulation of the gut-brain axis pathways with negligible toxicity, suggesting that NanoGABA is a promising therapeutic agent for major depressive disorders.

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.

Short-chain fatty acid-releasing nano-prodrugs for attenuating growth and metastasis of melanoma.

Low-molecular-weight (LMW) short-chain fatty acids (SCFAs), such as propionic and butyric acids, have been reported to possess anti-neoplastic effects; however, rapid renal clearance and high dose-based side effects limit their clinical translation. Hence, in this study, we have designed a new self-assembling nano-prodrugs that can effectively supply SCFAs: endogenous enzyme-metabolizable block copolymer poly(ethylene glycol)block-poly(vinyl ester) possessing several units of SCFAs conjugated as side chains via ester linkages. These amphiphilic polymers spontaneously self-assemble into nanostructures under aqueous conditions to form orally administrable nano-prodrugs (butyric acid: NanoBA and propionic acid: NanoPA). Herein, we show the therapeutic efficacy of SCFA nanoparticles (NanoSCFA) in a mouse model of metastasis (melanoma). Ad libitum intake of our NanoSCFA markedly demonstrated a decrease in the metastatic tumor nodules in the lungs compared with the effect observed after LMW SCFA administration with no discernible toxicity to the GI tract. In contrast, LMW SCFAs, even at a lower concentration than that of the NanoSCFA, facilitated villus atrophy. Taken together, our work suggests that the use of NanoSCFA as a therapeutic intervention for metastatic cancer is preferable over typical LMW SCFAs. STATEMENT OF SIGNIFICANCE: Low-molecular-weight (LMW) short-chain fatty acids (SCFAs) have shown versatile therapeutic effects on various diseases, including anti-tumorigenesis effects. However, their clinical translation is limited due to their poor pharmacokinetic profile and adverse effects. To overcome these limitations, we have developed new amphiphilic block copolymer-based SCFA-prodrugs, which self-assemble into nanoparticles in aqueous media (NanoSCFA). SCFAs are covalently conjugated to the hydrophobic polymer segment via ester linkage, which can be enzymatically metabolized after oral administration. In the present study, we confirmed that ad libitum intake of NanoSCFAs retarded the growth and metastatic potential of B16-F10 tumors compared to the LMW SCFAs with negligible discernible toxicity, reflecting NanoSCFA as a preferable therapeutic intervention to LMW SCFA counterparts.

Direct evidence for the involvement of intestinal reactive oxygen species in the progress of depression via the gut-brain axis.

Depression is a serious global social problem. Various therapeutic drugs have been developed based on the monoamine hypothesis; however, treatment-resistant depression is a common clinical issue. Recently, the gut-brain axis, which is associated with the hypothesis that the intestinal environment affects the brain, has garnered significant interest, and several studies have attempted to treat brain disorders based on this axis. These attempts include fecal transplantation, probiotics and prebiotics. In this study, we focused on intestinal reactive oxygen species (ROS) because excessive ROS levels disturb the intestinal environment. To elucidate the impact of scavenging intestinal ROS on depression treatment via the gut-brain axis, a novel polymer-based antioxidant (siSMAPoTN), which was distributed only in the intestine and did not diffuse into the whole body after oral administration, was used. siSMAPoTN selectively scavenged intestinal ROS and protected the intestinal environment from damage caused by chronic restraint stress (CRS). In addition, siSMAPoTN suppressed physiological and behavioral depression-related symptoms in the CRS mouse model.

Self-assembling polymer-based short chain fatty acid prodrugs ameliorate non-alcoholic steatohepatitis and liver fibrosis.

With the preponderance of a high-calorie diet and sedentary lifestyle, the prevalence of non-alcoholic steatohepatitis (NASH), a state of abnormally elevated lipid accumulation in the liver with chronic inflammation, is increasing at an alarming rate worldwide. Hence, cost-effective therapeutic interventions are required to manage this disease at an early stage. Numerous reports have suggested a link between gut microbial dysbiosis, particularly a decrease in the abundance of short-chain fatty acids (SCFA)-producing microbiota and NASH pathogenesis. Considering these low molecular weight (LMW) SCFAs such as acetic, propionic, and butyric acids have been used to inhibit hepatic steatosis in mouse models. However, the poor pharmacokinetic (PK) profile of SCFAs, caused due to their LMW, renders them therapeutically ineffective. Thus, to improve the PK characteristic-based therapeutic efficacy of LMW SCFAs, we designed SCFA-based prodrugs that possess self-assembling characteristics in aqueous media. The designed SCFA prodrugs consist of enzyme-metabolizable amphiphilic block copolymers, [poly(ethylene glycol)-b-poly(vinyl ester)s] conjugated to propionic acid (PA) or butyric acid (BA) by an ester linkage, which self-assemble into stable nanosized micelles several tens of nanometers in diameter (NanoPA and NanoBA). Via pharmacological analysis, we confirmed that, after oral administration, LMW BA decreased to a physiological level within 24 h in the liver, whereas BA liberated from NanoBA was observed until 72 h post-administration, implying a sustained release profile. Here, we evaluated the therapeutic efficacy of NanoSCFA in a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced NASH and liver fibrosis mouse model by ad libitum drinking. NanoSCFA, particularly NanoBA, exhibited the remarkable potential to ameliorate the phenotypic features of fatty liver disease by reducing hepatic lipogenesis and fibrosis, with negligible adverse effects. In contrast, conventional LMW SCFAs failed to prevent the pathogenesis of fatty liver disease, which plausibly can be explained by their rapid clearance and discernible adverse effects. Mechanistic studies revealed that NanoBA restored the nuclear expression of PPARα, a transcriptional factor regulating mitochondrial fatty acid oxidation, in the periportal hepatocytes and decreased the CPT1A expression level in the hepatic tissues, reflecting the therapeutic effects of NanoBA. Taken together, we confirmed that our NanoSCFA potentially improved the PK properties of SCFAs, and it consequently alleviated NASH symptoms and fibrotic liver compared to LMW SCFAs. Our study establishes NanoSCFA as a suitable nano-assembled prodrug for NASH treatment.

Design of a new self-assembling antioxidant nanomedicine to ameliorate oxidative stress in zebrafish embryos.

Oxidative stress, which is a persistent state of elevated reactive oxygen species (ROS), is implicated in the pathogeneses of several diseases, making antioxidant-based therapeutics the aptest intervention. Nevertheless, the clinical failure of conventional low-molecular-weight (LMW) antioxidants in oxidative stress-related diseases to yield favorable therapeutic outcomes and an increased mortality rate attributable to their poor pharmacokinetic characteristics, necessitates the development of alternative therapeutics. In light of this, we designed and synthesized a new amphiphilic polymer functionalized with a clinically safe base polymer of poly(styrene-co-maleic anhydride) copolymer conjugated with the LMW pleiotropic antioxidant TEMPO (a potent antioxidant) and biocompatible poly(ethylene glycol) (TEMPO-installed PSMA-g-PEG), which self-assembles into nano-sized micelles (SMAPoTN) under physiological conditions. We investigated its safety and antioxidant ability using zebrafish models. Common LMW antioxidants, such as 4-hydroxy-TEMPO (TEMPOL), vitamin C, N-acetyl-L-cysteine, and edaravone exposure induced phenotypic distortions, a manifestation of developmental toxicity, and resulted in high lethality in zebrafish larvae. LMW TEMPOL also adversely affected embryo hatchability, induced arrhythmia and cardiac edema, and failed to protect against oxidative stress. In contrast, exposure of zebrafish embryos to SMAPoTN increased the hatchability, protected embryos against various inducers of oxidative stress, and did not induce any phenotypic alterations or discernible toxicity. Taken together, we conclude that SMAPoTN surpasses LMW TEMPOL in terms of the ability to protect zebrafish, attributable to efficient ROS scavenging without perturbing normal redox homeostasis. These results imply that SMAPoTN can be used as a therapeutic intervention against various oxidative stress-induced diseases. STATEMENT OF SIGNIFICANCE: Failure of low molecular weight (LMW) antioxidants to improve therapeutic index in various oxidative stress-related pathogenesis, attributable to their poor pharmacokinetic characteristics, greatly limits their clinical translation. To overcome this limitation, we developed a self-assembling antioxidant nanoparticle (SMAPoTN) comprised of amphiphilic polymer; poly(styrene-co-maleic anhydride) conjugated with TEMPO as an antioxidant and biocompatible poly(ethylene glycol). Preliminary studies carried out in the in vivo models of zebrafish embryos confirmed that exposure of LMW antioxidant resulted in acute developmental toxicity, high lethality, and failure to rescue embryos against oxidative stress inducers. In contrast, SMAPoTN did not exert discernible toxicity and significantly improved their survival under oxidative stress. Our finding establishes antioxidant nanoparticles as more suitable therapeutic intervention for oxidative stress-induced diseases than LMW antioxidants.

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.

Antioxidative Self-Assembling Nanoparticles Attenuate the Development of Steatohepatitis and Inhibit Hepatocarcinogenesis in Mice.

Oxidative stress (OS) contributes to nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. We investigated whether antioxidative self-assembling nanoparticles (SMAPoTN) could reduce the development of NASH and hepatocellular carcinoma (HCC) in p62/Sqstm1 and Nrf2 double knockout (DKO) mice and studied protective mechanisms. We measured disease development in male DKO mice fed a normal chow (NASH model) or a 60% high-fat diet (HFD; HCC model) with or without SMAPoTN administration for 26 weeks. SMAPoTN inhibited liver fibrosis in both groups and prevented HCC development (0% vs. 33%, p < 0.05) in the HFD group. SMAPoTN reduced OS, inflammatory cytokine signaling, and liver fibrosis. RNA-sequencing revealed that SMAPoTN decreased endoplasmic reticulum stress signaling genes in both groups, HCC driver genes, and cancer pathway genes, especially PI3K-AKT in the HFD groups. In the SMAPoTN treatment HFD group, serum lipopolysaccharide levels and liver lipopolysaccharide-binding protein expression were significantly lower compared with those in the nontreatment group. SMAPoTN improved the α-diversity of gut microbiota, and changed the microbiota composition. Oral SMAPoTN administration attenuated NASH development and suppressed hepatocarcinogenesis in DKO mice by improving endoplasmic reticulum stress in the liver and intestinal microbiota. SMAPoTN may be a new therapeutic option for NASH subjects and those with a high HCC risk.

Design of self-assembling anti-epileptic drug for long-acting drug delivery in vivo.

Valproic acid (VPA) has been extensively used for the treatment of seizures in epilepsy. The recommended VPA concentration in the blood is in the range of 50-100 µg mL-1 and its therapeutic efficiency is well recognized. Since its therapeutic range is relatively narrow, strict scheduling of daily self-medication is required to optimize therapeutic outcomes and avoid adverse effects. To facilitate patient convenience in long-term and chronic therapies, the development of a sustained drug delivery system for VPA is a promising strategy. In this study, an enzyme-metabolizable block copolymer possessing a valproate ester, poly(ethylene glycol)-b-poly(vinyl valproate), was synthesized. The synthesized block copolymers formed stable nanoparticles (denoted NanoVPA) by self-assembly under physiological conditions and released VPA via enzymatic hydrolysis. NanoVPA showed improved pharmacokinetics compared to sodium valproate in vivo, and therapeutic efficacy in a pentylenetetrazol (PTZ)-induced kindling mouse model after once-weekly administration.

Sorafenib-loaded silica-containing redox nanoparticles for oral anti-liver fibrosis therapy.

Liver fibrosis is a chronic disease resulting from repetitive or prolonged liver injury with limited treatment options. Sorafenib has been reported to be a potential antifibrotic agent; however, its therapeutic effect is restricted because of its low bioavailability and severe adverse effects in the gastrointestinal (GI) tract. In this study, we developed sorafenib-loaded silica-containing redox nanoparticles (sora@siRNP) as an oral nanomedicine to treat liver fibrosis. The designed siRNP were prepared by self-assembly of amphiphilic block copolymers, which possess antioxidant nitroxide radicals as a side chain of the hydrophobic segment and porous silica particles in the nanoparticle core. The silica moieties in the core formed a crosslink between the self-assembling block copolymers to afford stable drug absorption, which could be useful in harsh GI conditions after oral drug administration. Based on in vitro evaluation, sora@siRNP exerted antiproliferative and antifibrotic effects against hepatic stellate cells (HSCs) and low toxicity against normal endothelial cells. A pharmacokinetic study showed that siRNP significantly improved the bioavailability and distribution of sorafenib in the liver. In an in vivo study using a mouse model of CCl4-induced liver fibrosis, oral administration of sora@siRNP significantly suppressed the fibrotic area in comparison to free sorafenib administration. In mice with CCl4-induced fibrosis, free sorafenib administration did not suppress the expression of α-smooth muscle actin; however, mice treated with sora@siRNP showed significantly suppressed expression of α-smooth muscle actin, indicating the inhibition of HSC activation, which was confirmed by in vitro experiments. Moreover, oral administration of free sorafenib induced severe intestinal damage and increased leakage into the gut, which can be attributed to the generation of reactive oxygen species (ROS). Our antioxidant nanocarriers, siRNP, reduced the adverse effects of local ROS scavenging in the GI tract. Our results suggest that sora@siRNP could serve as a promising oral nanomedicine for liver fibrosis.

A silica-based antioxidant nanoparticle for oral delivery of Camptothecin which reduces intestinal side effects while improving drug efficacy for colon cancer treatment.

Camptothecin (CPT) is a potent anticancer agent for the treatment of colorectal cancer; however, it exhibits some limitations, including poor solubility, low stability, and low bioavailability via oral administration, which restrict its usability in clinical treatments. In addition, overproduction of reactive oxygen species (ROS) during chemotherapy induces drug resistance and severe intestinal side effects. In this study, silica-installed ROS scavenging nanoparticles (siRNP) with 50-60 nm in diameter were employed to overcome the aforementioned drawbacks of CPT. The solubility of CPT was significantly improved by incorporating it into the core of the nanoparticle, forming CPT-loaded siRNP (CPT@siRNP). The anticancer activity of CPT@siRNP against colorectal cancer cells (C-26) in vitro was significantly improved as compared to free CPT through higher efficiency of intracellular internalization and induction of apoptosis. Owing to its antioxidant properties, CPT@siRNP reduced cytotoxicity to normal endothelial cells, which was in sharp contrast to the high toxicity of free CPT. Oral administration of CPT and CPT@siRNP to the C-26 tumor-bearing mice exhibited antitumor activity, accompanied by effective suppression of tumor growth. Although CPT treatment suppressed tumor progression, it caused severe side effects, including intestinal damage and significant bodyweight loss. Interestingly, such noticeable side effects were not observed in the mice treated with CPT@siRNP, and the effect of tumor growth inhibition tended to be similar to or higher than that of CPT treatment. The results obtained in this study indicate that CPT@siRNP is a potential therapeutic nanomedicine for the treatment of colon cancer. STATEMENT OF SIGNIFICANCE: Here we employed silica-containing antioxidant nanoparticle (siRNP) as promising oral delivery nanocarrier of campothecin (CPT) to treat colon cancer. The design of siRNP via covalent conjugation of antioxidant nitroxide radicals and the silanol groups in the polymer backbone contributes to a significant increase in the absorption of hydrophobic drug molecules inside the core and enhances the stability of nanoparticles in the gastrointestinal environment for oral drug delivery. CPT-loaded siRNP (CPT@siRNP) significantly improved solubility of CPT. As compared to free CTP, the CPT@siRNP treatment showed a significantly higher toxicity to colon cancer cell, inhibition of cancer cell migration, and induction of apopotosis. With the antioxidant feature, siRNP also significantly suppressed the intestinal side effects caused by CPT treatment in tumor-bearing mouse model.

Self-Assembling Antioxidants for Ischemia-Reperfusion Injuries.

Significance: Ischemia-reperfusion (IR) injury is a major component of severe damage in vascular occlusion during stroke, myocardial infarction, surgery, and organ transplantation, and is exacerbated by the excessive generation of reactive oxygen species (ROS), which occurs particularly during reperfusion. With the aging of the population, IR injury is becoming a serious problem in various organs, such as the kidney, brain, and heart, as well as in the mesenteric capillaries. Recent Advances: To prevent reperfusion injuries, natural and synthetic low-molecular-weight (LMW) antioxidants have been well studied. Critical Issues: However, these LMW antioxidants have various problems, including adverse effects due to excessive cellular uptake and their rapid clearance by the kidney, and cannot fully exert their potent antioxidant capacity in vivo. Future Directions: To overcome these problems, we designed and developed redox polymers with antioxidants covalently conjugated with them. These polymers self-assemble into nanoparticles in aqueous media, referred to as redox nanoparticles (RNPs). RNPs suppress their uptake into normal cells, accumulate at inflammation sites, and effectively scavenge ROS in damaged tissues. We had developed two types of RNPs: RNPN, which disintegrates in response to acidic pH; and RNPO, which does not collapse, regardless of the environmental pH. Utilizing the pH-sensitive and -insensitive characteristics of RNPN and RNPO, respectively, RNPs were found to exhibit remarkable therapeutic effects on various oxidative stress disorders, including IR injuries. Thus, RNPs are promising nanomedicines for use as next-generation antioxidants. This review summarizes the therapeutic impacts of RNPs in the treatment of kidney, cerebral, myocardial, and intestinal IR injuries. Antioxid. Redox Signal. 36, 70-80.

Design of enzyme-responsive short-chain fatty acid-based self-assembling drug for alleviation of type 2 diabetes mellitus.

Short-chain fatty acids (SCFAs), such as propionic and butyric acids have been touted as potential therapeutic interventions that can ameliorate diabetic pathogenesis. However, SCFAs are low-molecular-weight (LMW) compounds that have limited clinical use due to unfavorable pharmacokinetics, off-target effects, poor palatability and unpleasant odor. Hence, to improve the therapeutic utilization of SCFAs, the enzyme metabolizable block copolymers, [poly(ethylene glycol)-b-poly(vinyl ester)s], possessing propionate and butyrate esters were synthesized, which formed stable nanoparticles by self-assembling under physiological conditions. In this study, the therapeutic efficacy of propionic acid- and butyric acid-based self-assembling nanoparticles (PNP/BNP) was evaluated in a mouse model of type 2 diabetes mellitus through ad libitum drinking. The conventional antidiabetic drug, exenatide- and BNP-treated mice showed the highest glucose tolerance, whereas LMW SCFAs remained ineffective in normalizing glucose homeostasis. The better efficacy of BNP over the LMW SCFAs was attributable to (i) higher consumption of BNP than the LMW SCFAs by the mice (good palatability and odorless), (ii) prolonged residence time of BNP (48 h) in the gastro-intestinal tract (muco-adhesion) contributing to intestinal enzyme-mediated sustained release of butyric acid, and (iii) negligible off-target effects (no abrupt rise in the bloodstream). The aforementioned data suggest that SCFA-based nanoparticles are more potential therapeutic interventions than LMW SCFAs for metabolic diseases such as diabetes.

Antioxidant Nanomedicine Significantly Enhances the Survival Benefit of Radiation Cancer Therapy by Mitigating Oxidative Stress-Induced Side Effects.

Oxidative stress-induced off-target effects limit the therapeutic window of radiation therapy. Although many antioxidants have been evaluated as radioprotective agents, none of them are in widespread clinical use, owing to the side effects of the antioxidants themselves and the lack of apparent benefit. Aiming for a truly effective radioprotective agent in radiation cancer therapy, the performance of a self-assembling antioxidant nanoparticle (herein denoted as redox nanoparticle; RNP) is evaluated in the local irradiation of a subcutaneous tumor-bearing mouse model. Since RNP is covered with a biocompatible shell layer and possesses a core-shell type structure of several tens of nanometers in size, its lifetime in the systemic circulation is prolonged. Moreover, since 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), one of the most potent antioxidants, is covalently encapsulated in the core of RNP, it exerts intense antioxidant activity and induces fewer adverse effects by avoiding leakage of the TEMPO molecules. Preadministration of RNP to the mouse model effectively mitigates side effects in normal tissues and significantly extends the survival benefit of radiation cancer therapy. Moreover, RNP pretreatment noticeably increases the apoptosis/necrosis ratio of radiation-induced cell death, a highly desirable property to reduce the chronic side effects of ionizing irradiation.