When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

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A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers in vivo, can be used for targeting. Here, we show that nanoparticles can acquire a selective targeting capability with a protein corona adsorbed on the surface. We demonstrate that lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction with human plasma components, spontaneously become coated with vitronectin that promotes efficient uptake in cancer cells expressing high levels of the vitronectin ανβ3 integrin receptor.

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum 'Ofanto'). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4-7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed. © 2008 Springer-Verlag

Flavonoids have been recognised as one of the largest and most widespread groups of plant secondary metabolites, with marked antioxidant properties. The general name flavonoid refers to a class of more than 6500 molecules based upon a 15-carbon skeleton. In this paper a general overview of flavonoids, their classification, structures and analytical methods for their determination is presented.

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a 'protein corona'. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.

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