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.
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of heme catabolism and has been assumed to be important in cellular response against oxidative stress through modification of the pro-oxidant heme into less toxic catabolites that behave as antioxidants. However, the precise mechanisms involved and the physiological significance of such activity remain to be clarified. To elucidate roles HO-1 plays in vivo, hepatocyte-specific conditional knockout (CKO) mice of HO-1 gene were generated by site-specific recombination using albumin-promoter-driven Cre-loxP system. In livers of HO-1 CKO mice HO-1 protein level decreased to approximately 30% of control mouse livers. The HO-1 CKO mice are viable, exhibit normal growth curves over six months, and show no histological and serological abnormalities. We found that several cytoprotective genes, such as NAD(P)H dehydrogenase quinone 1 and glutathione S-transferase P1, showed markedly elevated expression, suggesting the increase of oxidative stress in HO-1 CKO mice even under quiescent conditions. In vivo electron paramagnetic resonance studies demonstrated that signal decay times of nitroxyl radicals were significantly longer in livers of HO-1 CKO mice than that of control mice, indicating that radical scavenging activity was significantly compromised in the mutant liver. HO-1 CKO mice were susceptible to carbon tetrachloride hepatotoxicity. These results provide the first in vivo evidence that HO-1 acts to protect cells against the oxidative stress in both basal conditions and upon chemical insult.heme oxygenase-1; oxidative stress; reactive oxygen species; in vivo EPR; carbon tetrachloride. Tohoku
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