1 Journal of Nanoscience Letters J. Nanosci. Lett. 2013, 3: 28 Cognizure www.cognizure.com/pubs © Cognizure. All rights reserved. Uptake of quantum dots into a freshwater flatworm: Intracellular accumulation and transmission from parents to offspring Laetitia De Jong a,†,* , Xavier Moreau a,† , Isabelle Bestel b,c , Emmanuel Beaudoin d , Ahissan Aimé b,c , Christel Dolain b,c , Gladys Saez a , Alain Tonetto e , Philippe Barthélémy b,c , Alain Thiéry a,† a IMBE UMR-CNRS 7263 - IRD 237, Aix-Marseille University, France b INSERM U869, Bordeaux, F-33076, France c University of Bordeaux, Bordeaux, F-33076, France d UMR 7273 - Institut de Chimie Radicalaire, Aix-Marseille University, France e Fédération de Chimie, Pôle PRATIM, Aix-Marseille University, France † Members of the International Consortium for the Environmental Implications of NanoTechnology-iCEINT & C’Nano-Provence-Alpes-Côte d’Azur * Author for correspondence: Laetitia De Jong, email: laetitia.dejong-moreau@imbe.fr Received 29 Oct 2012; Accepted 15 Nov 2012; Available Online 15 Nov 2012 1. Introduction Among the many examples of investigated nanomaterials, fluorescent semi-conductor nanocrystals known as quantum dots (QDs) are emerging as one of the most promising nanosystems [1, 2] for various scientific domains such as microelectronic [3], biology [4] or medicine [5]. In biological field, newly synthesized QDs are functionalized by secondary coatings to improve water solubility, core durability and suspension characteristics that make these nanoparticles biocompatible [6]. In parallel to these ever-increasing QD-based nanotechnologies and to the logically expected large production of QDs, ecotoxicological investigations are required to assess the effects of QD exposure on the aquatic environment, particularly since it receives run-off and wastewater from both domestic and industrial sources [2]. To date, a limited number of studies are tending to evaluate the acute toxicity or fate of QDs on a few aquatic invertebrates, either used in ecotoxicity tests such as cnidarians, Hydra vulgaris [7], crustaceans, Ceriodaphnia dubia and Daphnia magna or bivalves [8]. The purpose of this study is to examine the in vivo uptake of functionalized CdSe/ZnS core-shell QDs on a new freshwater invertebrate model: the Mesostoma lingua flatworm. This pond water species was previously used to investigate the in vivo uptake of an amphiphilic copolymer [9]. Aiming at investigating the environmental risk of engineered QDs, a suspension of aggregated functionalized QDs was prepared to mimic actual contaminations observed after the stagnation into pond waters. The bioavailability and fate of functionalized QDs within flatworm cells, ultrastructural cell damages and the transfer of QD-micelles from tissues of exposed adults into eggs are presented. 2. Experimental Details 2.1. Preparation of aggregated functionalized QD formulation CdSe/ZnS Core-shell QDs (red emitting-620 nm, Evidot ® ) covered by TOPO (tri-n-octylphosphine) (10 mg.mL -1 in toluene) was purchased from Evident Technologies (USA). The QD concentration, provided by the manufacturer, was used to estimate an absorption coefficient of aqueous QD concentrations. An amount of 10 μL (0.25 nmol) of this solution was added in a flask and dried under vacuum during 30 min for toluene evaporation. QDs were then suspended in 1 mL of chloroform with 25 nmol (100 eq) of DOPC (1,2-DiOleoyl-sn-glycéro-3-PhosphoCholine, purchased from Bachem ® ) and 25 nmol (100 eq) of DOTAU (N-[5’-(2’,3’-DiOleoyl) Uridine]-N’,N’,N’-TriméthylAmmon- ium Tosylate) [10]. The resulting mixture was dried to form a film in a round-bottom flask. Water soluble QD micelles were formed by hydrating the lipidic film in 1 mL of MilliQ water and bath sonication for 10 min at 60°C. Empty micelles were removed with centrifugation at 14 000 rpm for 10 min. The pellet containing aggregated and non aggregated QD-micelles were re-suspended in water and stored at 4°C in the dark. The Abstract The in vivo uptake of functionalized nanoplatform, Quantum Dots (QD: CdSe/ZnS) encapsulated with nucleolipids, were studied in freshwater platyhelminths, Mesostoma lingua. Nanoplatform bioavailability, fate within flatworms and ultrastructural damages were investigated using small angle X-ray scattering investigations and both confocal and transmission electron microscopies. We report QD accumulation in cytoplasm and organelles (cilia, vesicles, endoplasmic reticulum, Golgi bodies, mitochondria and nucleus) of different cell types as function of time exposure (24 h to 96 h) and concentrations (from 0.25 to 1 000 μg.L -1 ). A relevant result is the transfer of QDs from adult tissues to offspring. In spite of the presence of an embryonic shell, transmission electronic microscopy observations evidence a failure of this natural barrier to protect embryos from QD internalization and raise the question of the long term consequences to the offsprings. Keywords: Ecotoxicity; Aquatic invertebrates; Nucleolipids; Nanoparticles; Quantum Dots