The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

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Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.

. To make a PEGylated recombinant immunotoxin with improved therapeutic properties, we prepared a mutant of antiTac(Fv)-PE38 (LMB-2), a recombinant immunotoxin composed of a single-chain Fv fragment of the anti-human Tac monoclonal antibody to the IL-2 receptor ␣ subunit fused to a 38-kDa fragment of Pseudomonas exotoxin. For site-specific PEGylation of LMB-2, one cysteine residue was introduced into the peptide connector (ASGCGPE) between the Fv and the toxin. This mutant LMB-2 (cys1-LMB-2), which retained full cytotoxic activity, was then sitespecifically conjugated with 5 or 20 kDa of polyethylene glycol-maleimide. When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar cytotoxic activities in vitro but superior stability at 37°C in mouse serum, a 5-to 8-fold increase in plasma half-lives in mice, and a 3-to 4-fold increase in antitumor activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity. Site-specific PEGylation of recombinant immunotoxins may increase their therapeutic potency in humans.Pseudomonas exotoxin ͉ cancer therapy ͉ immunotherapy ͉ pharmacokinetics ͉ liver toxicity

BackgroundClarifying the physicochemical properties of nanomaterials is crucial for hazard assessment and the safe application of these substances. With this in mind, we analyzed the relationship between particle size and the in vitro effect of amorphous nanosilica (nSP). Specifically, we evaluated the relationship between particle size of nSP and the in vitro biological effects using human keratinocyte cells (HaCaT).ResultsOur results indicate that exposure to nSP of 70 nm diameter (nSP70) induced an elevated level of reactive oxygen species (ROS), leading to DNA damage. A markedly reduced response was observed using submicron-sized silica particles of 300 and 1000 nm diameter. In addition, cytochalasin D-treatment reduced nSP70-mediated ROS generation and DNA damage, suggesting that endocytosis is involved in nSP70-mediated cellular effects.ConclusionsThus, particle size affects amorphous silica-induced ROS generation and DNA damage of HaCaT cells. We believe clarification of the endocytosis pathway of nSP will provide useful information for hazard assessment as well as the design of safer forms of nSPs.

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