Gamma-Glutamylcysteine Production Using Phytochelatin Synthase-Like Enzyme Derived from Nostoc sp. Covalently Immobilized on a Cellulose Carrier.

Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer's disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications.

Subvisible Particles Derived by Dropping Stress Enhance Anti-PEG Antibody Production and Clearance of PEGylated Proteins in Mice.

Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs.

Chitosan nanoparticles and their Tween 80 modified counterparts disrupt the developmental profile of zebrafish embryos.

Chitosan nanoparticles (CS-NPs) and their Tween 80 modified counterparts (TmCS-NPs) are among the most commonly used brain-targeted vehicles. However, their potential developmental toxicity is poorly understood. In this study, zebrafish embryos are introduced as an in vivo platform. Both NPs showed a dose-dependent increase in developmental toxicity (decreased hatching rate, increased mortality and incidences of malformation). Neurobehavioral changes included decreased spontaneous movement in TmCS-NP treated embryos and hyperactive effect in CS-NP treated larvae. Both NPs remarkably inhibited axonal development of primary and secondary motor neurons, and affected the muscle structure. Overall, this study demonstrated that CS-NPs and TmCS-NPs could affect embryonic development, disrupt neurobehavior of zebrafish larvae and affect muscle and neuron development, suggesting more attention on biodegradable chitosan nanoparticles.

Effect of amorphous silica nanoparticles on in vitro RANKL-induced osteoclast differentiation in murine macrophages.

Amorphous silica nanoparticles (nSP) have been used as a polishing agent and/or as a remineralization promoter for teeth in the oral care field. The present study investigates the effects of nSP on osteoclast differentiation and the relationship between particle size and these effects. Our results revealed that nSP exerted higher cytotoxicity in macrophage cells compared with submicron-sized silica particles. However, tartrate-resistant acid phosphatase (TRAP) activity and the number of osteoclast cells (TRAP-positive multinucleated cells) were not changed by nSP treatment in the presence of receptor activator of nuclear factor κB ligand (RANKL) at doses that did not induce cytotoxicity by silica particles. These results indicated that nSP did not cause differentiation of osteoclasts. Collectively, the results suggested that nanosilica exerts no effect on RANKL-induced osteoclast differentiation of RAW264.7 cells, although a detailed mechanistic examination of the nSP70-mediated cytotoxic effect is needed.

Therapeutic effect of PEGylated TNFR1-selective antagonistic mutant TNF in experimental autoimmune encephalomyelitis mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease, the pathogenesis of which is related to elevated serum levels of tumor necrosis factor-α (TNF). Although anti-TNF therapy has been tested as a potential treatment for MS, no remission of symptoms was observed. Recent reports indicated that the TNFR1 signal was responsible for the pathogenesis of murine experimental autoimmune encephalomyelitis (EAE), while the TNFR2 signal was responsible for recovery of the pathogenesis of EAE. Therefore, selective blocking of TNFR1 appears to be a promising strategy for the treatment of MS. In this regard, we previously succeeded in developing a novel TNFR1-selective antagonistic TNF mutant (R1antTNF) by using phage display technology. Here, we have examined the therapeutic potential of R1antTNF using EAE mice. Treatment with PEGylated R1antTNF (PEG-R1antTNF) significantly improved the clinical score and cerebral demyelination at the onset of EAE. Considerable suppression of Th1 and Th17-type response was also observed in spleen and lymph node cells of mice given PEG-R1antTNF. Moreover, the administration of PEG-R1antTNF suppressed the infiltration of inflammatory cells containing Th1 and Th17 cells into the spinal cord. These results suggest that selective blocking of TNFR1 by PEG-R1antTNF could be an effective therapeutic strategy against MS.