pH-Sensitive Radical-Containing-Nanoparticle (RNP) for the L-Band-EPR Imaging of Low pH Circumstances

Abstract

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.

Novel redox nanomedicine improves gene expression of polyion complex vector

“…Before the effect of RNP on the transfection efficiency was evaluated, the interaction between RNP and the PEI/pDNA polyplex was investigated by electron spin resonance (ESR). We have already confirmed that the ESR spectra of RNP markedly change from a broad to sharp triplet due to the disintegration of RNP [20]. If RNP interacts with the PEI/pDNA polyplex, a change in the ESR spectra of RNP must be observed owing to the morphology change in RNP.…”

“…RNP was prepared following the methods of Yoshitomi et al [20]. An amphiphilic block copolymer possessing a poly(ethylene glycol) (PEG) chain as a hydrophilic segment and a polystyrene chain as a hydrophobic segment with TEMPO repeating units was prepared using two-step reactions.…”

“…After 24 h, PEI/pDNA complex (DNA = 0.75 µg per well, N/P = 10), RNP (final polymer concentrations were 0.033, 0.33, 3.3 and 33 µM), and TNF-α (final concentrations were 0.06, 0.6 and 6 nM) were added to each well. The RNP concentration which is low enough not to induce cytotoxicity was used according to our previous paper [20]. After a 24 h incubation, the transfection medium was changed to fresh DMEM with 10% FBS, and the cells were further incubated for 24 h. After the incubation, the cells were washed with phosphate-buffered saline (PBS) twice.…”

“…We have developed a novel nitroxide radical-containing nanoparticle (RNP) that effectively scavenges ROS because it contains 3000 to 5000 nitroxide radicals in the core (figure 1) [20,21]. The RNP was prepared from self-assembling amphiphilic block copolymers (MeO-PEG-b-PMS-TEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(methylstyrene) (PMS) segment possessing TEMPO moieties.…”

Creation of a blood-compatible surface: A novel strategy for suppressing blood activation and coagulation using a nitroxide radical-containing polymer with reactive oxygen species scavenging activity

“…Core-shell-type polymeric micelles containing nitroxide radicals have already been developed for the treatment of oxidative stress injuries and in vivo ESR imaging [29,30]. It has been confirmed that they not only strongly scavenge ROS in vivo or in vitro but also significantly prevent oxidative stress damage in an cerebral ischemia-reperfusion injury model in rats [31], renal ischemia-reperfusion injury model in mice [32] and a neuron cell line used as a model for Alzheimer's disease [33,34], in which excess ROS is generated.…”