RESUMO
The large amount of cauliflower industry waste represents an unexplored source of bioactive compounds. In this work, peptide hydrolysates from cauliflower leaves were characterized by combined bioanalytical approaches. Twelve peptide fractions were studied to evaluate unexplored biological activities by effect-based cellular bioassays. A potent inhibition of intracellular xanthine oxidase activity was observed in human vascular endothelial cells treated with one fraction, with an IC50 = 8.3 ± 0.6 µg/ml. A different fraction significantly induced the antioxidant enzyme superoxide dismutase 1 and decreased the tumor necrosis factor α-induced VCAM-1 expression, thus leading to a significant improvement in the viability of human vascular endothelial cells. Shotgun peptidomics and bioinformatics were used to retrieve the most probable bioactive peptide sequences. Our study shows that peptides from cauliflower waste should be recycled for producing valuable products useful for the prevention of endothelial dysfunction linked to atherogenesis progression.
Assuntos
Brassica/química , Peptídeos/uso terapêutico , Xantina Oxidase/química , Células Endoteliais , Humanos , Peptídeos/farmacologiaRESUMO
Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.
Assuntos
Apolipoproteínas/química , Endocitose , Lipídeos , Nanopartículas/metabolismo , Coroa de Proteína , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Pinocitose , Distribuição TecidualRESUMO
The self-assembling processes underlining the capabilities of facially differentiated ("Janus") polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications.
Assuntos
Ciclodextrinas/química , DNA/química , Coroa de Proteína/química , Transfecção/métodos , Biofísica , Células HeLa , Humanos , Células MCF-7 , NanopartículasRESUMO
When nanoparticles come into contact with biological media, they are covered by a biomolecular 'corona', which confers a new identity to the particles. In all the studies reported so far nanoparticles are incubated with isolated plasma or serum that are used as a model for protein adsorption. Anyway, bodily fluids are dynamic in nature so the question arises on whether the incubation protocol, i.e. dynamic vs. static incubation, could affect the composition and structure of the biomolecular corona. Here we let multicomponent liposomes interact with fetal bovine serum (FBS) both statically and dynamically, i.e. in contact with circulating FBS (≈40 cm s(-1)). The structure and composition of the liposome-protein corona, as determined by dynamic light scattering, electrophoretic light scattering and liquid chromatography tandem mass spectrometry, were found to be dependent on the incubation protocol. Specifically, following dynamic exposure to FBS, multicomponent liposomes were less enriched in complement proteins and appreciably more enriched in apolipoproteins and acute phase proteins (e.g. alpha-1-antitrypsin and inter-alpha-trypsin inhibitor heavy chain H3) that are involved in relevant interactions between nanoparticles and living systems. Supported by our results, we speculate that efficient predictive modeling of nanoparticle behavior in vivo will require accurate knowledge of nanoparticle-specific protein fingerprints in circulating biological media.
Assuntos
Nanopartículas/química , Coroa de Proteína/análise , Animais , Apolipoproteínas/química , Bovinos , Cromatografia Líquida de Alta Pressão , Difusão Dinâmica da Luz , Lipossomos/química , Proteômica , Espectrometria de Massas em TandemRESUMO
Here we introduce a proteomics methodology based on nanoliquid-chromatography tandem mass spectrometry (nanoLC/MS-MS) to investigate the "protein corona effect for targeted drug delivery", an innovative strategy, which exploits the "protein corona" that forms around nanoparticles in a physiological environment to target cells.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Cromatografia Líquida/métodos , Lipossomos/farmacologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Lipossomos/metabolismo , Polietilenoglicóis/química , Adulto JovemRESUMO
As soon as nanomaterials, such as nanoparticles (NPs), are injected into a physiological environment a rich coating of biomolecules known as the "protein corona" rapidly covers them. This protein dress is the main factor, which affects the interaction of NPs with living systems. While the relationship between NP features and the biomolecule corona has been extensively investigated, whether and how changes in the physiological environment affect the NP-protein corona remains under-investigated. This is one of the most important steps in translating results in animal models to the clinic. Here we investigated thoroughly the biological identity of lipid NPs (size, charge, aggregation state and composition of the corona) after incubation with human plasma (HP) and mouse plasma (MP) by dynamic light scattering, micro-electrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC/MS-MS). Specifically, we used two different liposomal formulations: the first one was made of polyethyleneglycol (PEG)-coated 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), while the second one was made of 30% of DOTAP, 50% of neutral saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 20% cholesterol. The temporal evolution and complexity of the NP-protein corona was found to be strongly dependent on the biological environment. In MP, liposomes were more negatively charged, less enriched in opsonins and appreciably more enriched in apolipoproteins than their counterparts in HP. Collectively, our results suggest that the biological identities of NPs in mice and humans can be markedly different from each other. Relevance of results to in vivo applications is discussed.