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.

The ultimate objective of nanoparticle-based therapy is to functionalize nanomedicines in a micro-disease environment without any side effects. Here, we reveal that our pH-responsive nitroxide radical-containing nanoparticles (RNP(pH)) disintegrate within the renal acidic lesion and act as scavengers of reactive oxygen species (ROS), leading to a relief of acute kidney injury (AKI). RNP(pH) was prepared using amphiphilic block copolymers possessing 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moieties via amine linkage as a side chain of the hydrophobic segment. The self-assembled RNP(pH) disintegrated at pH below 7.0 because of a protonation of the amino groups in the hydrophobic core of the nanoparticles, thereby resulting in an improvement in ROS scavenging activity. Using a renal ischemia-reperfusion AKI model in mice, the therapeutic effect of RNP(pH) on ROS damage was evaluated. Unlike the RNP without pH-triggered disintegration (RNP(Non-pH)), the RNP(pH) showed extremely high ROS scavenging activity and renal protective effects. It is interesting to note that the side effect of nitroxide radicals was markedly suppressed due to the compartmentalization of nitroxide radicals in the core of RNP(pH) in untargeted area. The morphology changes in RNP(pH) were confirmed by analyzing electron spin resonance spectra, and these findings provide the evidence of the real therapeutic effect of the environment-sensitive specific disintegration of nanoparticles in vivo.

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.

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.