báo cáo khoa học: "The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture" pps

Santos et al Journal of Nanobiotechnology 2010, 8:24 http://www.jnanobiotechnology.com/content/8/1/24 RESEARCH Open Access The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture Ana R Santos1,2*, Ana S Miguel1,3, Leonor Tomaz2, Rui Malhó4, Christopher Maycock3,4, Maria C Vaz Patto2, Pedro Fevereiro2,4, Abel Oliva1 Abstract Background: Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility Results: A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation Conclusions: Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications Background Nanotechnology is a fast-developing industry, having substantial impact on the economy, society and the environment [1] and predictions so far exceed the Industrial Revolution, with a $1 trillion market by 2015 [2] Nanotechnology has the potential to revolutionize the agricultural and food industry with new tools for the molecular treatment of diseases, rapid disease detection and enhancing plant ability to absorb nutrients Smart sensors and smart delivery systems will help the * Correspondence: raquelsantos@itqb.unl.pt Biomolecular Diagnostics Laboratory, Instituto de Tecnologia Qmica e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal Full list of author information is available at the end of the article agricultural industry to fight viruses and other crop pathogens [3] However, the novel size-dependent properties of nanomaterials, that make them desirable in technical and commercial uses, also create concerns in terms of environmental and toxicological impact [4] Nanotoxicology is emerging as an important subdiscipline of nanotechnology and involves the study of the interactions of nanostructures with biological systems Nanotoxicology aims on elucidating the relationship between the physical and chemical properties of nanostructures with the induction of toxic biological responses [5] This information is important to characterize nanomaterial in biotechnology, ecosystems, agriculture and biomedical applications [6] © 2010 Santos et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Santos et al Journal of Nanobiotechnology 2010, 8:24 http://www.jnanobiotechnology.com/content/8/1/24 The few studies conducted to date on the effects of nanoparticles on plants have focused mainly on phytotoxicity and how certain plant metabolic functions are affected The reported effects vary depending on the type of nanoparticle, as well as plant species, and are inconsistent among studies [2] So far, there is only one report of nanoparticle toxicity in cells of a photosynthetic organism, the green microalgae Chlamydomonas reinhardtii, in which the toxicity of two types of widely used nanomaterials (TiO2 and CdTe) was evaluated [7] No data is available concerning toxicology of Quantum Dots (QDs) in higher plant cells [8] QDs are inorganic semiconductor nanocrystals, typically composed of a cadmium selenide (CdSe) core and a zinc sulphide (ZnS) shell and whose excitons (excited electron-holepairs) are confined in all three dimensions, giving rise to characteristic fluorescent properties QDs are extremely photostable, bright and are characterized by broad absorption profiles, high extinction coefficients and narrow and spectrally tunable emission profiles [9] Cell-based in vitro studies play an essential role on meaningful toxicity testing They allow the setting up of high-throughput systems for rapid and cost-effective screening of hazards, while targeting the biological responses under highly controlled conditions [4] The evaluation of five categories of cellular response, including reactive oxygen species (ROS) production and accumulation, cell viability, cell stress, cell morphology, and cellparticle uptake, are central themes in such testing [10] Aiming to develop a nano-strategy using coated QDs conjugated with specific biomolecules to precociously identify the presence of fungal infections in Medicago sativa (a perennial pulse with economic relevance) we established a fine plant cell suspension culture that was subsequently used to investigate the potential cytotoxicity of CdSe/ZnS Mercaptopropanoic acid coated QDs and its uptake at cellular level Methods Cell suspension culture establishment Cell suspension cultures were established from a Medicago sativa line M699, seeds being kindly provided by Diego Rubiales (IAS-CSIC, Spain) Well-developed petioles from 25 day old in vitro germinated M699 seedlings were used as explants for callus induction Petioles were placed in solid Murashige & Skoog (M&S) medium supplemented with 0.5 mg/L of 2.4-D and kinetin and mg/L of dithiothreitol, maintained in growth chamber under a 16 hours photoperiod and a day/night temperature of 24°/22°C (Phytotron Edpa 700, Aralab, Portugal) Two friable portions of weeks old dark grown callus from petioles were placed in a 250 mL Erlenmeyer flask with 50 mL of liquid M&S medium, supplemented with the same growth hormone composition used for callus Page of 14 phase The flasks were maintained in an orbital shaker at 110 rpm (Innova 4900, New Brunswick Scientific, Germany) in the dark, at 24°C After 10 days, 100 mL of fresh medium was added 10 days later, 100 mL of fresh medium was added to 20 mL of decanted cell suspension culture Until an adequate cell density was obtained, the cells were pelleted and medium replaced every 8/9 days When stabilized, suspensions were sub cultured every days transferring 20 mL to 100 mL of fresh medium (in 250 mL Erlenmeyer flasks) Growth regulators were always filter sterilized through 0.2 μm Orange Scientific filters and added to cooled autoclaved medium (20 minutes at 121°C).These cell suspension cultures will be referred to as stock Synthesis, solubilisation and characterization of CdSe/ZnS core shell QDs All chemicals unless indicated were obtained from Sigma-Aldrich and used as received UV-vis absorbance spectra were taken using a Beckman DU-70 Photoluminescence spectra were recorded with a SPEX Fluorolog spectrofluorimeter TOPO/HDA - capped CdSe nanocrystals were synthesized using standard procedures [11] This typically generates CdSe nanocrystals with the first absorption peak around 580-590 nm and a diameter of 3.6-4.5 nm For the synthesis of core-shell CdSe/ZnS QDs, the cores obtained were monodisperse after passivation with at least monolayers of ZnS Passivation was obtained using the SILAR method [12] that consists of alternating injections of Zn and S precursors to the solution containing CdSe-core nanocrystals suspended in octadecene/hexadecylamine After extraction with methanol, centrifugation and decantation, the particles were dispersed in chloroform for further processing The Mercaptopropanoic acid coated nanocrystals were synthesized by the phase transfer method as described previously [12] The obtained Mercaptopropanoic acid coated CdSe/ZnS QDs were then concentrated using a Sartorius Vivaspin tube (cutoff 10 KDa) at 7.500 g For the characterization of the synthesized CdSe/ZnS QD nanoparticles Transmission Electron Microscopy (TEM) was used Low resolution images were obtained using a JEOL 200CX traditional TEM operating at an acceleration voltage of 200 kV Quantum yields were measured relative to Rhodamine 6G with excitation at 530 nm Solutions of QDs in chloroform or water and dye in ethanol were optically matched at the excitation wavelength (l = 530 nm) Dynamic Light Scattering (DLS) analysis was performed using a Nano series dynamic light scatterer from Malvern With this equipment the hydrodynamic diameter (HD) and zeta potential (ξ) of synthetic CdSe/ZnS and their corresponding Mercaptopropanoic acid coated Santos et al Journal of Nanobiotechnology 2010, 8:24 http://www.jnanobiotechnology.com/content/8/1/24 particles were measured For the HD analyses all the samples were between 0.06 and 0.3 μM and filtered through a 0.2 μm filter before analyses HD were obtained from number-weighted size distribution analysis and reported as the mean of triplicate measurements ξ-Potential for Mercaptopropanoic acid coated QDs with concentration between 0.06 and 0.3 μM were measured in H2O Milli-Q basified to pH = 12 Values are reported as the average of triplicate runs consisting of 20 runs at 25°C Similarly, the ξ-potential of the mercaptopropanoic acid-QDs in M&S medium was measured using the DLS equipment Value reported is the average of triplicate runs consisting of 14 runs at 25°C Imaging and Microscopy settings Unless stated otherwise, images were acquired in a Nikon Eclipse TE2000-S (Japan) inverted microscope equipped with a HMX-4 100 W Mercury lamp and appropriate filter settings Images were acquired with an Evolution MP 5.1 megapixel digital CCD Color Camera (Media Cybernetics) controlled by Image Pro Plus 5.0 software (Media Cybernetics) Thin time-course confocal optical sections (~2 μm thick) were acquired with a Leica SP-E Confocal Laser Scanning Microscope using