Toru Yoshitomi

Design of core--shell-type nanoparticles carrying stable radicals in the core.

Utilizing the self-assembled core-shell-type polymeric micelle technique, high-performance nanoparticles possessing stable radicals in the core and reactive groups on the periphery were prepared. The anionic ring-opening polymerization of ethylene oxide (EO) was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)-methanesulfonate (acetal-PEG-Ms; 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl and acetal terminal groups (acetal-PEG-SH) (2) in a highly selective and quantitative manner. Poly(ethylene glycol)-block-poly(chloromethylstyrene) (acetal-PEG-b-PCMS) (3) was synthesized by the free-radical telomerization of chloromethylstyrene (CMS) using 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (4). The obtained 4 formed core-shell-type nanoparticles in aqueous media when subjected to the dialysis method: the cumulant average diameter of the nanoparticles was about 40 nm, and the nanoparticles emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid. This means that these nanoparticles are anticipated as high-performance bionanoparticles that can be used in vivo.

An Orally Administered Redox Nanoparticle That Accumulates in the Colonic Mucosa and Reduces Colitis in Mice

BACKGROUND & AIMS Drugs used to treat patients with ulcerative colitis are not always effective because of nonspecific distribution, metabolism in the gastrointestinal tract, and side effects. We designed a nitroxide radical-containing nanoparticle (RNP(O)) that accumulates specifically in the colon to suppress inflammation and reduce the undesirable side effects of nitroxide radicals. METHODS RNP(O) was synthesized by assembly of an amphiphilic block copolymer that contains stable nitroxide radicals in an ether-linked hydrophobic side chain. Biodistribution of RNP(O) in mice was determined from radioisotope and electron spin resonance measurements. The effects of RNP(O) were determined in mice with dextran sodium sulfate (DSS)-induced colitis and compared with those of low-molecular-weight drugs (4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl [TEMPOL] or mesalamine). RESULTS RNP(O), with a diameter of 40 nm and a shell of poly(ethylene glycol), had a significantly greater level of accumulation in the colonic mucosa than low-molecular-weight TEMPOL or polystyrene latex particles. RNP(O) was not absorbed into the bloodstream through the intestinal wall, despite its long-term retention in the colon, which prevented its distribution to other parts of the body. Mice with DSS-induced colitis had significantly lower disease activity index and less inflammation following 7 days of oral administration of RNP(O) compared with mice with DSS-induced colitis or mice given low-molecular-weight TEMPOL or mesalamine. CONCLUSIONS We designed an orally administered RNP(O) that accumulates specifically in the colons of mice with colitis and is more effective in reducing inflammation than low-molecular-weight TEMPOL or mesalamine. RNP(O) might be developed for treatment of patients with ulcerative colitis.

pH-sensitive radical-containing-nanoparticle (RNP) for the L-band-EPR imaging of low pH circumstances.

For the imaging of low pH circumstances in vivo, a pH-sensitive radical-containing-nanoparticle (RNP), which has an intense electron paramagnetic resonance (EPR) signal, was designed and developed using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO. This RNP formed core-shell-type micelles in the physiological environment, and the cumulant average diameter of the RNP was about 50 nm. The cytotoxicity and acute toxicity studies for the RNP revealed that the median inhibitory concentration (IC(50)) of TEMPO radicals in RNP core and median lethal dose (LD(50)) of RNP were >8 mmol N(TEMPO)/L and >600 mg/kg (>960 mumol N(TEMPO)/kg), respectively, indicating fairly low toxicity. The blood circulation of the RNP was evaluated using ICR mice. Contrary to the rapid clearance of low-molecular-weight TEMPO derivatives such as 4-hydroxy-TEMPO (TEMPOL) from the bloodstream, the EPR signal of the RNP remained for a fairly long period of time. Actually, the signal was observed in the blood for more than 2 h, as monitored by EPR spectroscopy. The compartmentalization of the TEMPO radicals in the RNP core improved the stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PCTEMPO segment, the disintegration of the RNP was caused by the protonation of the amino groups in response to the acidic pH environment (pH < 6.0), as confirmed by the dynamic light scattering (DLS) measurements. In addition, a drastic change in the EPR spectra from broad to sharp triplet was observed, accompanying the disintegration. This change was based upon the mobility of the TEMPO moieties covalently conjugated in the hydrophobic segment, which was confirmed by the rotational correlation time of the TEMPO moieties on the PCTEMPO segment. Note that the peak intensity of the EPR signal increased at around the phase transition point (ca. pH = 6.0). When pH-sensitive RNP solutions at pH values 5.6 and 7.4 were visualized using an L-band EPR imaging system, the phantom images showed a remarkable on-off regulation in response to the acidic pH environment. These results demonstrate that pH-sensitive RNPs are expected to serve as nanoprobes for the in vivo EPR imaging of low pH circumstances.

Newly Synthesized Radical-Containing Nanoparticles Enhance Neuroprotection After Cerebral Ischemia-Reperfusion Injury

BACKGROUND:Antioxidant nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) have been investigated for their ability to scavenge free radicals produced by ischemia-reperfusion injury. However, the short in vivo half-life and toxicity of TEMPO have limited their clinical application. OBJECTIVE:We developed a core-shell-type nanoparticle, termed a radical-containing nanoparticle (RNP), to deliver nitroxyl radicals with prolonged in vivo half-life and pH-sensitivity. We evaluated the ability of RNP to deliver TEMPO radicals to the ischemic brain and scavenge free radicals in cerebral ischemia-reperfusion injury using rats. METHODS:When RNPs were administrated to middle cerebral artery occlusion rats, the delivery and clearance of RNPs were detected using electron paramagnetic resonance (EPR) assay. The production of superoxide anion in neuronal cells was observed with dihydroethidium staining. The treatment effects were evaluated by measuring the cerebral infarction volumes, lipid peroxidation and protein oxidation, and neurological symptom scoring. RESULTS:The TEMPO radicals contained in RNPs were detected for 6 hours after intravenous administration as a triplet EPR signal in the ischemic brain, and RNPs significantly reduced the production of superoxide anion in neuronal cells compared with saline and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyls (TEMPOL). The infarction volumes of rats treated by RNPs were significantly lower than those of rats treated by saline, micelles, and TEMPOL. In addition, RNP treatment suppressed lipid peroxidation and protein oxidation, and limited the adverse effects of TEMPO radicals such as hypotension. CONCLUSION:RNPs could be a promising neuroprotective agent with their enhanced ability to scavenge free radicals and reduced toxicity.

Redox nanoparticle treatment protects against neurological deficit in focused ultrasound-induced intracerebral hemorrhage

BACKGROUND Intracerebral hemorrhage is reported to induce the generation of reactive oxygen species and oxidative DNA damage in the brain. AIMS We aimed to examine whether our designed redox polymer nanoparticle could reduce intracerebral hemorrhage induced by 1-MHz focused ultrasound sonication coupled with microbubble treatment. MATERIALS & METHODS Contrast-enhanced ultrasound imaging, frozen section, brain edema, neurologic deficit, the number of morphologically normal neurons, DNA oxidization and superoxide anion generation were used to investigate the neuroprotective effect of redox polymer nanoparticles. RESULTS We confirmed that the 1-MHz focused ultrasound coupled with microbubble produced intracerebral hemorrhage and showed that the redox polymer nanoparticle ameliorates intracerebral hemorrhage-induced brain edema, neurological deficit and oxidative damage. CONCLUSION These results suggest that redox polymer nanoparticle is a potential therapeutic agent for intracerebral hemorrhage induced by focused ultrasound.

Redox nanoparticle therapeutics to cancer increase in therapeutic effect of doxorubicin, suppressing its adverse effect

The ultimate goal of cancer chemotherapy is to achieve a cure without causing any adverse effects. We have developed a pH-sensitive redox nanoparticle (RNP(N)), which disintegrates under acidic conditions and exposes nitroxide radicals, leading to strongly scavenging reactive oxygen species (ROS). After intravenous administration of RNP(N) to tumor bearing mice, it effectively accumulated in tumors due to the leaky neovascular and immature lymphatic system and scavenged ROS, resulting in suppression of inflammation and activation of NF-кB, after disintegration of RNP(N) in the tumors. Pre-administration of RNP(N) prior to treatments with anticancer agents, doxorubicin, to tumor-bearing mice significantly suppressed the progression of tumor size, compared to low-molecular weight 4-hydroxy-TEMPO. Interestingly, the administration of RNP(N) suppressed adverse effects of doxorubicin to normal organs due to the scavenging ROS and suppression of inflammation, which was confirmed by reduction in lactate dehydrogenase and creatine phosphokinase activities in plasma. RNP(N) is thus anticipated as a novel and ideal adjuvant for cancer chemotherapy.

Development of an oral nanotherapeutics using redox nanoparticles for treatment of colitis-associated colon cancer.

Oral chemotherapy is the preferred treatment for colon cancer. However, this strategy faces many challenges, including instability in the gastrointestinal (GI) tract, insufficient bioavailability, low tumor targeting, and severe adverse effects. In this study, we designed a novel redox nanoparticle (RNP(O)) that is an ideal oral therapeutics for colitis-associated colon cancer treatment. RNP(O) possesses nitroxide radicals in the core, which act as reactive oxygen species (ROS) scavengers. Orally administered RNP(O) highly accumulated in colonic mucosa, and specifically internalized in cancer tissues, but less in normal tissues. Despite of long-term oral administration of RNP(O), no noticeable toxicities were observed in major organs of mice. Because RNP(O) effectively scavenged ROS, it significantly suppressed tumor growth after accumulation at tumor sites. Combination of RNP(O) with the conventional chemotherapy, irinotecan, led to remarkably improved therapeutic efficacy and effectively suppressed its adverse effects on GI tract. Therefore, RNP(O) is promising oral nanotherapeutics for cancer therapies.

Redox-active injectable gel using thermo-responsive nanoscale polyion complex flower micelle for noninvasive treatment of local inflammation

Reactive oxygen species (ROS) scavengers have not been widely used for treatment of local inflammatory reactions such as arthritis and periodontal disease because they are rapidly eliminated from the inflamed site, which results in a low therapeutic effect. Therefore, to enhance the local retention time of ROS scavengers, we developed a redox-active injectable gel (RIG) system by using poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PMNT-PEG-PMNT) triblock copolymer, which possesses ROS scavenging nitroxide radicals as side chains of the PMNT segment. Cationic PMNT segment in PMNT-PEG-PMNT forms polyion complexes with anionic poly(acrylic acid) (PAAc) to form a flower-like micelle (ca. 79 nm), which exhibits in situ thermo-irreversible gelation under physiological conditions. We confirmed the prolonged site-specific retention time of RIG by performing in vivo noninvasive electron spin resonance imaging and quantitative evaluation. In contrast to low-molecular-weight nitroxide radical compounds that disappeared from injection sites in less than 1h after subcutaneous injection, 40% of the RIG remained even at 3 days. We also found that RIG inhibits neutrophil infiltration and cytokine production, which leads to suppression of hyperalgesia. These findings indicate the potential of RIG as an innovative approach for treatment of local inflammation.

Nitroxyl radical-containing nanoparticles for novel nanomedicine against oxidative stress injury

This article discusses the preparation and characterization of pH-sensitive nitroxyl radical-containing nanoparticles (RNPs) possessing nitroxyl radicals in the core and reactive groups on the periphery, and its biomedical application. The RNPs prepared by a self-assembling amphiphilic block copolymers composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxyls (TEMPOs) via an amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO are initially described. The cumulant average diameter of an RNP is approximately 40 nm, and the RNP has intense electron paramagnetic resonance signals. RNPs show a prolonged blood circulation time by the compartmentalization of nitroxyl radicals into the hydrophobic core, and disintegrate in response to a low pH environment, such as ischemic tissue, resulting in effectively scavenging reactive oxygen species due to an exposure of nitroxyl radicals from the RNP core. Thus, the RNP prepared was found to be effective for cerebral ischemia-reperfusion injury. Therefore, RNPs are promising as high-performance therapeutic nanomedicine for oxidative stress injuries.

Reactive oxygen species-scavenging nanomedicines for the treatment of oxidative stress injuries.

This Progress Report describes a development of two types of reactive oxygen species (ROS)-scavenging nanomedicines for the treatment of oxidative stress injuries, referred to as pH-sensitive redox nanoparticle (RNP(N) ) and pH-insensitive redox nanoparticle (RNP(O) ), which are prepared by self-assembling amphiphilic block copolymers possessing nitroxide radicals as a side chain of hydrophobic segment via amine and ether linkages, respectively. Due to a protonation of amino groups in hydrophobic core, RNP(N) disintegrates in low pH environments such as ischemic, inflamed, and tumor tissues, resulting in increased ROS-scavenging activity because of the exposed nitroxide radicals from the core. Utilizing pH-responsiveness of RNP(N) , it shows remarkable therapeutic effects on oxidative stress injuries such as renal and cerebral ischemia-reperfusion injuries after intravenous administration. Moreover, RNP(N) shows an enhancement of the activity of anticancer drugs by suppression of activation of transcription factors in tumor due to the ROS scavenging. On the other hand, orally administered RNP(O) has notable characteristics such as preferential accumulation in mucosa and inflamed area of gastrointestinal tract and no uptake into blood stream. Based on these characters, RNP(O) shows a remarkable therapeutic effect for the gastrointestinal inflammation without any adverse effects. Thus, ROS-scavenging nanomedicines have therapeutic efficacy in numerous oxidative stress diseases.