Novel application of quantum dots-lectin conjugates for labeling zebrafish epidermis

SIGOT, VALERIA; PAULO CABRAL FILHO; BEATE SAEGESSER SANTOS; ADRIANA FONTES

Conferencia; 18th International Conference in Luminescence; 2017

The use of fluorescent nanocrystals so called Quantum Dots (QDs), for labeling membranes has been explored through different conjugation strategies to target specific receptors on the cell surface. These approaches have been successfully applied for tracking receptors at the membranes or in vesicles, mostly in cell cultures. The use of QDs for in vivo labeling of animal embryos has been limited to microinjection into Xenopus laevis eggs, or as contrast agent in zebrafish [1]. In zebrafish (Danio rerio), the embryonic epidermis develops before 24 hours? post fertilization as a first protective barrier for pathogens. Exogenous labeling of membranes with fluorescent QDs has not been previously reported in developing zebrafish. We aimed to explore the use of QDs CdTe (~4 nm) conjugated to the lectin concanavalin A (ConA) to target glycoconjugates on epidermal cells in vivo as well as in fixed embryos. Preformed complexes were prepared by adsorption according to Tenório et al. [2], and added by direct incubation or by microinjection at 100 nM QDs. In living embryos, QDs-ConA labeling was observed as a dot pattern distribution along cells membranes (Fig. 1 C, D). The labeling, although specific was most prominent in head with the presence of dispersed aggregates and no homogeneous labeling along the whole embryo. No labeling was observed after incubation with free QDs (Fig. 1, E, F). In addition, no evident toxicity was observed under the conditions tested. As expected, after fixation and permeabilization a homogeneous membrane labeling of the whole embryo was observed (panel A, B) either with free QDs or QDs-ConA conjugates. These observations suggest that in vivo the skin acts as a protective barrier being impermeable to nanoparticles but still accessible for specific QDs-ConA binding. As a novel observation, unconjugated QDs revealed label labyrinth like patterns known as actin microridges which are present on mucosal surfaces across animal species, including the surface epithelium of zebrafish larvae.