Nanobody-siRNA Conjugates for Targeted Delivery of siRNA to Cancer Cells.

Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.

Methionine stimulates motor impairment and cerebellar mercury deposition in methylmercury-exposed mice.

Methylmercury (MeHg) is a highly toxic environmental contaminant that produces neurological and developmental impairments in animals and humans. Although its neurotoxic properties have been widely reported, the molecular mechanisms by which MeHg enters the cells and exerts toxicity are not yet completely understood. Taking into account that MeHg is found mostly bound to sulfhydryl-containing molecules such as cysteine in the environment and based on the fact that the MeHg-cysteine complex (MeHg-S-Cys) can be transported via the L-type neutral amino acid carrier transport (LAT) system, the potential beneficial effects of L-methionine (L-Met, a well known LAT substrate) against MeHg (administrated as MeHg-S-Cys)-induced neurotoxicity in mice were investigated. Mice were exposed to MeHg (daily subcutaneous injections of MeHg-S-Cys, 10 mg Hg/kg) and/or L-Met (daily intraperitoneal injections, 250 mg/kg) for 10 consecutive days. After treatments, the measured hallmarks of toxicity were mostly based on behavioral parameters related to motor performance, as well as biochemical parameters related to the cerebellar antioxidant glutathione (GSH) system. MeHg significantly decreased motor activity (open-field test) and impaired motor performance (rota-rod task) compared with controls, as well as producing disturbances in the cerebellar antioxidant GSH system. Interestingly, L-Met administration did not protect against MeHg-induced behavioral and cerebellar changes, but rather increased motor impairments in animals exposed to MeHg. In agreement with this observation, cerebellar levels of mercury (Hg) were higher in animals exposed to MeHg plus L-Met compared to those only exposed to MeHg. However, this event was not observed in kidney and liver. These results are the first to demonstrate that L-Met enhances cerebellar deposition of Hg in mice exposed to MeHg and that this higher deposition may be responsible for the greater motor impairment observed in mice simultaneously exposed to MeHg and L-Met.

Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake.

Various forms of mercury possess different rates of absorption, metabolism and excretion, and consequently, toxicity. Methylmercury (MeHg) is a highly neurotoxic organic mercurial. Human exposure is mostly due to ingestion of contaminated fish. Ethylmercury (EtHg), another organic mercury compound, has received significant toxicological attention due to its presence in thimerosal-containing vaccines. This study was designed to compare the toxicities induced by MeHg and EtHg, as well as by their complexes with cysteine (MeHg-S-Cys and EtHg-S-Cys) in the C6 rat glioma cell line. MeHg and EtHg caused significant (p<0.0001) decreases in cellular viability when cells were treated during 30min with each mercurial following by a washing period of 24h (EC50 values of 4.83 and 5.05µM, respectively). Significant cytotoxicity (p<0.0001) was also observed when cells were treated under the same conditions with MeHg-S-Cys and EtHg-S-Cys, but the respective EC50 values were significantly increased (11.2 and 9.37µM). l-Methionine, a substrate for the l-type neutral amino acid carrier transport (LAT) system, significantly protected against the toxicities induced by both complexes (MeHg-S-Cys and EtHg-S-Cys). However, no protective effects of l-methionine were observed against MeHg and EtHg toxicities. Corroborating these findings, l-methionine significantly decreased mercurial uptake when cells were exposed to MeHg-S-Cys (p=0.028) and EtHg-S-Cys (p=0.023), but not to MeHg and EtHg. These results indicate that the uptake of MeHg-S-Cys and EtHg-S-Cys into C6 cells is mediated, at least in part, through the LAT system, but MeHg and EtHg enter C6 cells by mechanisms other than LAT system.

Probucol increases striatal glutathione peroxidase activity and protects against 3-nitropropionic acid-induced pro-oxidative damage in rats.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD involve oxidative stress and mitochondrial dysfunction. Administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of the mitochondrial enzyme succinate dehydrogenase, in rodents has been proposed as a useful experimental model of HD. This study evaluated the effects of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, on the biochemical parameters related to oxidative stress, as well as on the behavioral parameters related to motor function in an in vivo HD model based on 3-NP intoxication in rats. Animals were treated with 3.5 mg/kg of probucol in drinking water daily for 2 months and, subsequently, received 3-NP (25 mg/kg i.p.) once a day for 6 days. At the end of the treatments, 3-NP-treated animals showed a significant decrease in body weight, which corresponded with impairment on motor ability, inhibition of mitochondrial complex II activity and oxidative stress in the striatum. Probucol, which did not rescue complex II inhibition, protected against behavioral and striatal biochemical changes induced by 3-NP, attenuating 3-NP-induced motor impairments and striatal oxidative stress. Importantly, probucol was able to increase activity of glutathione peroxidase (GPx), an enzyme important in mediating the detoxification of peroxides in the central nervous system. The major finding of this study was that probucol protected against 3-NP-induced behavioral and striatal biochemical changes without affecting 3-NP-induced mitochondrial complex II inhibition, indicating that long-term probucol treatment resulted in an increased resistance against neurotoxic events (i.e., increased oxidative damage) secondary to mitochondrial dysfunction. These data appeared to be of great relevance when extrapolated to human neurodegenerative processes involving mitochondrial dysfunction and indicates that GPx is an important molecular target involved in the beneficial effects of probucol.

Effects of platelet-activating factor on the interaction of Trypanosoma cruzi with Rhodnius prolixus.

We investigated the effects of platelet-activating factor (PAF) on the interaction of Trypanosoma cruzi with Rhodnius prolixus. The parasites (epimastigotes) were treated with PAF and/or WEB 2086 (PAF antagonist) for 1 h prior to the interaction experiments. PAF stimulated both in vivo and ex vivo interactions between T. cruzi and R. prolixus while WEB 2086 abrogated these effects. PAF-treated epimastigotes also showed an increase in surface negativity and in the amount of surface sialic acid. Neither of these effects was observed when the epimastigotes were treated with neuraminidase following PAF treatment. In the ex vivo interaction experiments, the number of epimastigotes bound to the midguts of the insects was reduced when the epimastigotes had been treated with neuraminidase. We conclude that PAF modulates the interaction of T. cruzi with R. prolixus by altering the amount of sialyl residues at the surface of the parasite.

Development of a ligand blot assay using biotinylated live cells.

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.