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Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Biological functions of cell membranes and their correlation to the heterogeneity of the latter%s lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes. Biološke funkcije celičnih membran in njihove korelacije s heterogenostjo njihove lipidne sestave so še vedno slabo raziskane. Fluorescenca omogoča enega izmed najbolj vsestranskih pristopov k raziskovanju bioloških membran, vendar je za označevanje plazemskih membran na voljo le malo svetlih in fotostabilnih fluorescenčnih označevalcev. Načrtovali in sintetizirali smo dve novi tovrstni fluorescenečni barvili, spojini 8 in 9, ki temeljita na tiazolo-kumarinskem ogrodju. Obe izkazujeta občutljivost na okolje in kažeta podobne premike v emisijskih spektrih v različnih topilih kot je bilo opaženo pri 7-nitrobenz-2-oksa-1,3-diazol-4-ilu (NBD). Drugo, pozitivno nabito barvilo 9, obarva plazemsko membrano selektivno in z omejenim prerazporejanjem v ostale celične membrane. Žal pa 9, za razliko od preostalih dveh testiranih barvil, torej 8 in 6-NBD-PC, zelo hitro fotobledi. Ne glede na to, razvoj dveh novih tiazolo-kumarinskih membranskih barvil predstavlja smiselen pristop k načrtovanju novih membranskih barvil.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. They are best investigated using non-invasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labelling of nanoparticles with fluorescent dyes. When performed inadequately, unbound fluorophores and inadvertently altered chemical and physical properties of the nanoparticles can, however, result in experimental artefacts and erroneous conclusions.To prevent such unintentional errors, we here describe a minimal combination of experimental methods to enable artefact-free fluorescent labelling of metal-oxide nanoparticles – the largest subpopulation of nanoparticles by industrial production and applications – and demonstrate its application in the case of TiO2 nanotubes. We 1) characterize potential changes of the nanoparticles’ surface charge and morphology that might occur during labelling, and 2) assess stable binding of the fluorescent dye to nanomaterial, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials’ mechanism of action and will foster widespread and safe use of new nanomaterials.Abstract Figure

Background & aimTiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.Methods & resultsFluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.ConclusionThese results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.