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The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery are currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC-liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, a cell proliferation and death study along with the MTT test on HeLa cells treated with NC-liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by the cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. In contrast, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and their carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as an optical probe for future in vivo experiments.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30 W and 20 kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32 min in detergent micelles, ranged from 3.8 to 6.5 min in the different proteoliposomes formulations, and 5.5 min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution.

Surface chemical composition of atmospheric particles plays an important role in determining reactivity and optical properties of aerosol than influencing its role in climate forcing (Ramanathan et al., 2001) and in the human health effects (Klejnowski et al. 2012, Kendall et al., 2001). In addition, surface composition is strongly depending on the sources/formation processes and on the size of particles. Particle modification in terms of composition and structure may also lead to multiple evolution pathways. There are many methods and instruments available for studying different properties of the surface of airborne Particulate Matter (PM), in terms of morphological and elemental composition (Coury and Dillner, 2008; Kirchner et al., 2003). X-ray photoelectron spectroscopy (XPS) could be a suitable technique to simultaneously investigate surface composition of particles and chemical speciation of the main elements like for example C, S, N, Na, Cl, Ca, Si, Mg and other minor hetero-elements. In this communication we present a systematic XPS surface study of different size fractions of PM. The samples analysed have been collected in a background site in Lecce inside the University Campus at about 4 km SW of the Lecce town (SE of Italy, coordinates 40°20'N, 18°06'E), in modality "size-segregated", using a 10-stage cascade impactor with rotating collection plates having an inlet with a nominal cut-off at 18 mm and 10 stages (MOUDI II, 120R). Flow-rate: 30 l/min and 10 stages (S1 - S10) nominal 50% efficiency cut-off at 10, 5.6, 3.2, 1.8, 1.0, 0.56, 0.32, Collection time was 48 h. Ten series of samples (100 sampling substrates in total) were collected between February and June 2011 on Al substrates (47 mm disks). The averaged mass size distribution showed a bimodal shape were the Accumulation mode was characterized with an of PM10) and the Coarse mode with an MMD=4.4 ± 0.4 44 ± 4.7% del PM10). XPS analysis has been focused first on the systematic evaluation of surface chemical composition of different size-segregated particles, to investigate the variability of the chemical surface composition comparing the accumulation and the coarse modes of atmospheric particles, and on the comparison between the chemical information (element quantitative distribution and speciation) of the surface with bulk chemical composition of collected particles when useful to investigate the generation processes and the sources of collected particles. The high resolution XPS spectra allowed to distinguish different organic functional groups (C-C/C=C, -C-O, -C=O/-C(O)N, -C(O)O, CO3=) and to speciate the detectable hetero-elements, sulphur (SO4=, sulphone and sulphide compounds), nitrogen (NH4+, NO3-, NO2- and organic-nitrogen compounds), sodium (Na+) and chlorine (Cl-) species. Significant differences in particles belonging to accumulation and coarse modes were observed and correlated with the formation processes and the sources from which particles originated. The oxygen concentrations was inversely correlated with carbon species concentrations, however, the content of oxidized organic carbon was not correlated with O content confirming that the O increment in coarse particles can be attributed to inorganic species (crustal origin) like CO3=, SiO2, Fe2O3, CaO, and NO3-. The speciation of nitrogen showed ammonium only in the accumulation mode and nitrate only in coarse mode excluding the presence of ammonium nitrate in the area studied. The SO4= was segregated in accumulation mode, moreover sulphide and sulphone are present only in the coarse mode . Based on these results, SO4= was associated to secondary aerosol form: (NH4)2SO4 and (NH4)HSO4. The occurrence of sulphone in coarse particles suggested the possibility of SOx incorporation into organic matter via photochemical processes on the surface of coarse mode. Results showed a correlation of Na+ and Cl- with larger abundances on the surface of large particles due to particles of marine origin. An excess of chloride was observed on the surface contrarily to the typical depletion observed in bulk concentrations. The two processes were correlated with larger depletions of chloride in the bulk associated, on average, with larger enrichments on the surface. Carbonate was present in the coarse fraction and it was associated with crustal aerosol.