Alois Bonifacio

Functionalized gold nanoparticles: a detailed in vivo multimodal microscopic brain distribution study

In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex.

Surface-enhanced Raman spectroscopy of blood plasma and serum using Ag and Au nanoparticles: a systematic study

Surface-enhanced Raman spectroscopy (SERS) is a good candidate for the development of fast and easy-to-use diagnostic tools, possibly used on biofluids in point-of-care or screening tests. In particular, label-free SERS spectra of blood serum and plasma, two biofluids widely used in diagnostics, could be used as a metabolic fingerprinting approach for biomarker discovery. This study aims at a systematic evaluation of SERS spectra of blood serum and plasma, using various Ag and Au aqueous colloids, as SERS substrates, in combination with three excitation lasers of different wavelengths, ranging from the visible to the near-infrared. The analysis of the SERS spectra collected from 20 healthy subjects under a variety of experimental conditions revealed that intense and repeatable spectra are quickly obtained only if proteins are filtered out from samples, and an excitation in the near-infrared is used in combination with Ag colloids. Moreover, common plasma anticoagulants such as EDTA and citrate are found to interfere with SERS spectra; accordingly, filtered serum or heparin plasma are the samples of choice, having identical SERS spectra. Most bands observed in SERS spectra of these biofluids are assigned to uric acid, a metabolite whose blood concentration depends on factors such as sex, age, therapeutic treatments, and various pathological conditions, suggesting that, even when the right experimental conditions are chosen, great care must be taken in designing studies with the purpose of developing diagnostic tests.

Label-free surface-enhanced Raman spectroscopy of biofluids: fundamental aspects and diagnostic applications

In clinical practice, one objective is to obtain diagnostic information while minimizing the invasiveness of the tests and the pain for the patients. To this end, tests based on the interaction of light with readily available biofluids including blood, urine, or saliva are highly desirable. In this review we examine the state of the art regarding the use of surface-enhanced Raman spectroscopy (SERS) to investigate biofluids, focusing on diagnostic applications. First, a critical evaluation of the experimental aspects involved in the collection of SERS spectra is presented; different substrate types are introduced, with a clear distinction between colloidal and non-colloidal metal nanostructures. Then the effect of the excitation wavelength is discussed, along with anomalous bands and artifacts which might affect SERS spectra of biofluids. The central part of the review examines the literature available on the SERS spectra of blood, plasma, serum, urine, saliva, tears, and semen. Finally, diagnostic applications are critically discussed in the context of the published evidence; this section clearly reveals that SERS of biofluids is most promising as a rapid, cheap, and non-invasive tool for mass screening for cancer.

Poly-l-lysine-Coated Silver Nanoparticles as Positively Charged Substrates for Surface-Enhanced Raman Scattering

Positively charged nanoparticles to be used as substrates for surface-enhanced Raman scattering (SERS) were prepared by coating citrate-reduced silver nanoparticles with the cationic polymer poly-l-lysine. The average diameter of the coated nanoparticles is 75 nm, and their zeta potential is +62.3 ± 1.7 mV. UV-vis spectrophotometry and dynamic light scattering measurements show that no aggregation occurs during the coating process. As an example of their application, the so-obtained positively charged coated particles were employed to detect nanomolar concentrations of the anionic chromophore bilirubin using SERS. Because of their opposite charge, bilirubin molecules interact with the coated nanoparticles, allowing SERS detection. The SERS intensity increases linearly with concentration in a range from 10 to 200 nM, allowing quantitative analysis of bilirubin aqueous solutions.

Polyol Synthesis of Silver Nanoparticles: Mechanism of Reduction by Alditol Bearing Polysaccharides

Alditol bearing chitosans have shown the ability to reduce silver ions in mild conditions and without addition of exogenous reducing agents. The ion reduction induces the formation of a lactone moiety on the polysaccharide (Fetizon reaction) without causing C-C bond cleavage on the polyol. The close and multivalent arrangement of the endogenous reducing agent (alditols) on the polysaccharide backbone resulted in the formation of silver nanoparticles (phi < 10 nm), which induced a considerable SERS effect and led to hydrogel formation.

New Insights on Cytological and Metabolic Features of Ostreopsis cf. ovata Fukuyo (Dinophyceae): A Multidisciplinary Approach

The harmful dinoflagellate Ostreopsis cf. ovata has been causing toxic events along the Mediterranean coasts and other temperate and tropical areas, with increasing frequency during the last decade. Despite many studies, important biological features of this species are still poorly known. An integrated study, using different microscopy and molecular techniques, Raman microspectroscopy and high resolution liquid chromatography-mass spectrometry (HR LC-MS), was undertaken to elucidate cytological aspects, and identify main metabolites including toxins. The species was genetically identified as O. cf. ovata, Atlantic-Mediterranean clade. The ultrastructural results show unique features of the mucilage network abundantly produced by this species to colonize benthic substrates, with a new role of trichocysts, never described before. The amorphous polysaccharidic component of mucilage appears to derive from pusule fibrous material and mucocysts. In all stages of growth, the cells show an abundant production of lipids. Different developmental stages of chloroplasts are found in the peripheral cytoplasm and in the centre of cell. In vivo Raman microspectroscopy confirms the presence of the carotenoid peridinin in O. cf. ovata, and detects in several specimen the abundant presence of unsaturated lipids structurally related to docosahexaenoic acid. The HR LC-MS analysis reveals that ovatoxin-a is the predominant toxin, together with decreasing amounts of ovatoxin-b, -d/e, -c and putative palytoxin. Toxins concentration on a per cell basis increases from exponential to senescent phase. The results suggest that benthic blooms of this species are probably related to features such as the ability to create a unique mucilaginous sheath covering the sea bottom, associated with the production of potent toxins as palytoxin-like compounds. In this way, O. cf. ovata may be able to rapidly colonize benthic substrates outcompeting other species.

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt‐Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455–461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV–VIS spectroscopy, as well as coupling efficiency and substrate‐binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3. Proteins 2008.

Spatial distribution of heme species in erythrocytes infected with Plasmodium falciparum by use of resonance Raman imaging and multivariate analysis.

The multivariate algorithm hierarchical cluster analysis is applied to sets of resonance Raman spectra collected from human erythrocytes infected with the malaria parasite Plasmodium falciparum. The images obtained yield information about the distribution of hemoglobin and hemozoin (or malaria pigment) within the parasitized cells and about their molecular structure. This method has the advantage of conveying more information than other imaging approaches based on resonance Raman spectroscopy, and it is a promising tool to study the hemozoin formation process and its interaction with antimalarial drugs within unstained, well-preserved parasites.

Enhanced oral bioavailability of vinpocetine through mechanochemical salt formation: physico-chemical characterization and in vivo studies.

ABSTRACTPurposeEnhancing oral bioavailability of vinpocetine by forming its amorphous citrate salt through a solvent-free mechanochemical process, in presence of micronised crospovidone and citric acid.MethodsThe impact of formulation and process variables (amount of polymer and citric acid, and milling time) on vinpocetine solubilization kinetics from the coground was studied through an experimental design. The best performing samples were characterized by employing a multidisciplinary approach, involving Differential scanning calorimetry, X-ray diffraction, Raman imaging/spectroscopy, X-ray photoelectron spectroscopy, solid-state NMR spectroscopy, porosimetry and in vivo studies on rats to ascertain the salt formation, their solid-state characteristics and oral bioavailability in comparison to vinpocetine citrate salt (Oxopocetine®).ResultsThe analyses attested that the mechanochemical process is a viable way to produce in absence of solvents vinpocetine citrate salt in an amorphous state.ConclusionFrom the in vivo studies on rats the obtained salt was four times more bioavailable than its physical mixture and bioequivalent to the commercial salt produced by conventional synthetic process implying the use of solvent.

Towards long lasting zirconia-based composites for dental implants: Transformation induced plasticity and its consequence on ceramic reliability

Zirconia-based composites were developed through an innovative processing route able to tune compositional and microstructural features very precisely. Fully-dense ceria-stabilized zirconia ceramics (84vol% Ce-TZP) containing equiaxed alumina (8vol%Al2O3) and elongated strontium hexa-aluminate (8vol% SrAl12O19) second phases were obtained by conventional sintering. This work deals with the effect of the zirconia stabilization degree (CeO2 in the range 10.0-11.5mol%) on the transformability and mechanical properties of Ce-TZP-Al2O3-SrAl12O19 materials. Vickers hardness, biaxial flexural strength and Single-edge V-notched beam tests revealed a strong influence of ceria content on the mechanical properties. Composites with 11.0mol% CeO2 or above exhibited the classical behaviour of brittle ceramics, with no apparent plasticity and very low strain to failure. On the contrary, composites with 10.5mol% CeO2 or less showed large transformation-induced plasticity and almost no dispersion in strength data. Materials with 10.5mol% of ceria showed the highest values in terms of biaxial bending strength (up to 1.1GPa) and fracture toughness (>10MPa√m). In these ceramics, as zirconia transformation precedes failure, the Weibull modulus was exceptionally high and reached a value of 60, which is in the range typically reported for metals. The results achieved demonstrate the high potential of using these new strong, tough and stable zirconia-based composites in structural biomedical applications. STATEMENT OF SIGNIFICANCE Yttria-stabilized (Y-TZP) zirconia ceramics are increasingly used for developing metal-free restorations and dental implants. Despite their success related to their excellent mechanical resistance, Y-TZP can undergo Low Temperature Degradation which could be responsible for restoration damage or even worst the failure of the implant. Current research is focusing on strategies to improve the LTD resistance of Y-TZP or to develop alternative composites with better stability in vivo. In this work the mechanical characterization of a new type of very-stable zirconia-based composites is presented. These materials are composed of ceria-stabilized zirconia (84vol%Ce-TZP) containing two second phases (α-alumina and strontium hexa-aluminate) and exhibit exceptional strength, toughness and ductility, which may allow the processing of dental implants with a perfect reliability and longer lifetime.