Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies† Patrizio Candeloro, * a Luca Tirinato, a Natalia Malara, a Annalisa Fregola, a Eudald Casals, b Victor Puntes, b Gerardo Perozziello, a Francesco Gentile, ac Maria Laura Coluccio, ac Gobind Das, c Carlo Liberale, c Francesco De Angelis c and Enzo Di Fabrizio ac Received 14th April 2011, Accepted 11th August 2011 DOI: 10.1039/c1an15313g Microinjection techniques and Raman spectroscopy have been combined to provide a new methodology to investigate the cytotoxic effects due to the interaction of nanomaterials with cells. In the present work, this novel technique has been used to investigate the effects of Ag and Fe 3 O 4 nanoparticles on Hela cells. The nano- particles are microinjected inside the cells and these latter ones are probed by means of Raman spectroscopy after a short incubation time, in order to highlight the first and impulsive mechanisms developed by the cells to counteract the presence of the nano- particles. The results put in evidence a different behaviour of the cells treated with nanoparticles in comparison with the control cells; these differences are supposed to be generated by an emerging oxidative stress due to the nanoparticles. The achieved results demonstrate the suitability of the proposed method as a new tool for nanotoxicity studies. Introduction Nanoparticles (NPs) and nanomaterials in general have been attracting the interest of a wide scientific community in the last decade, because of their novel properties with respect to bulk mate- rials. Thanks to their unique features, nanomaterials have been exploited in several application fields (such as energy, sensors, biomedical, and electronics), and more than 800 commercial prod- ucts based on nanomaterials were already available in 2008. 1 Many nanotechnology applications in life sciences, ranging from cell biology to medical diagnostics and therapeutics, clearly involve an interaction between nanomaterials and living systems. Former works have proposed the use of NPs as agents for drug delivery and ther- apeutics, 2–5 transfection vectors, 6 contrast agents for magnetic resonance imaging, 7 probes for spectroscopy 8 and fluorescent labels for microscopy. 9 Consequently, nanomaterials biocompatibility and toxicity are crucial points to be addressed for developing nanotech- nology products for biology and medicine. Recently a lot of effort has been devoted to investigate these issues for carbon nanotubes, 10–13 quantum dots, 14–16 and metal and oxide NPs. 17–20 However the role played by nanomaterials in cell physiology is still debated. This lack of clarity arises from the large number of parameters involved in nanotoxicology studies: in principle NP effects on cellular life could depend on the size, shape and chemical composition of the NPs, and identical NPs could have different effects on different cell lines. Furthermore it is now well accepted in the biomedical community that protein decoration of NPs plays a crucial role in the endocytosis process. 21–23 As soon as NPs are placed in physiological environments (including cell culturing medium) their high surface reactivity could make them completely decorated at the surface with a protein layer. At this point the interaction with cells, i.e. the rate and amount of endocytosis, is mainly driven from the biological interaction between the cell membrane receptors and the proteins of the NP decorations. As examples, both Chithrani et al. in ref. 22 and Jiang et al. in ref. 23 report a size-dependent uptake for spherical gold NPs, with the maximum around 50 nm diameter size. Smaller NPs, which are intuitively expected to penetrate more easily the cell membrane, are instead less efficiently phagocytized via receptor-mediated endocy- tosis. Since many nanotoxicology studies are carried out by incu- bating the NPs inside the cell culturing medium, it turns out that a full comprehension of experimental results could be puzzled by compli- cated endocytosis pathways due to proteins binding to NPs. In this work microinjection techniques are exploited to inject NPs directly inside the cells, without any residence time in the culturing medium. Hence, this novel approach avoids any protein decoration that could occur before the cellular uptake and eliminates any possible effect related to receptor-mediated endocytosis. Further- more, the interaction between cells and bare NPs is achieved directly inside the cellular system, and the overall cellular response will not be affected by the presence of proteins bound to NPs prior to the uptake. Since not all the cells from the same culturing dish can be micro- injected (as they are more than several thousands), an analytical tool at the single cell level has to be chosen for investigating the cellular response. In the present case the microinjected cells are probed by a BioNEM Laboratory, Experimental and Clinical Medicine Department, University ‘‘Magna Graecia’’ of Catanzaro, 88100 Loc. Germaneto, Catanzaro, Italy b Institut Catal  a de Nanotecnologia and Institut Catal  a de Recerca i Estudis Avanc ¸ats (ICREA), Barcelona, Spain c Italian Institute of Technology (IIT), Nanostructure Department, Via Morego 30, 16163 Genova, Italy † Electronic supplementary information (ESI) available. See DOI: 10.1039/c1an15313g This journal is ª The Royal Society of Chemistry 2011 Analyst Dynamic Article Links C < Analyst Cite this: DOI: 10.1039/c1an15313g www.rsc.org/analyst COMMUNICATION Downloaded by UNIVERSITA DI GENOVA - CCSBA on 30 August 2011 Published on 30 August 2011 on http://pubs.rsc.org | doi:10.1039/C1AN15313G View Online