Chelating Surfaces for Oriented Human Serum Albumin Molecules.

Protein immobilization in a specific conformation or orientation at an interface is influenced by specific interactions with the outer layer of the surface. A strategy to build-up a complex construct which is able to orient protein molecules, based on metal-cation chelation processes, is reported. The proposed methodology implies the formation of a mercaptoundecanoic acid monolayer on a gold surface that is activated to attach covalently the tripeptide glycyl-l-histidyl-l-lysine (GHK) on the surface, whose sites are then employed to chelate copper ions, providing a selective platform for the orientation of human serum albumin (HSA) molecules. The protein adsorption process on GHK and GHK-Cu(II)-complex surfaces was monitored by the in situ quartz crystal microbalance with dissipation monitoring (QCM-D) and force spectroscopy technique. The changes in frequency and dissipation factor as well as the D- f plots from QCM-D measurements help to characterize the changes in the protein conformation and are confirmed by force curve spectroscopy results. An improved kinetic model, based on random sequential adsorption with variable protein footprints, has been developed to predict and simulate the experimentally found HSA average surface coverage onto the GHK and GHK-Cu(II)-complex surfaces.

Preventing corona effects: multiphosphonic acid poly(ethylene glycol) copolymers for stable stealth iron oxide nanoparticles.

When dispersed in biological fluids, engineered nanoparticles are selectively coated with proteins, resulting in the formation of a protein corona. It is suggested that the protein corona is critical in regulating the conditions of entry into the cytoplasm of living cells. Recent reports describe this phenomenon as ubiquitous and independent of the nature of the particle. For nanomedicine applications, however, there is a need to design advanced and cost-effective coatings that are resistant to protein adsorption and that increase the biodistribution in vivo. In this study, phosphonic acid poly(ethylene glycol) copolymers were synthesized and used to coat iron oxide particles. The copolymer composition was optimized to provide simple and scalable protocols as well as long-term stability in culture media. It is shown that polymers with multiple phosphonic acid functionalities and PEG chains outperform other types of coating, including ligands, polyelectrolytes, and carboxylic acid functionalized PEG. PEGylated particles exhibit moreover exceptional low cellular uptake, of the order of 100 femtograms of iron per cell. The present approach demonstrates that the surface chemistry of engineered particles is a key parameter in the interactions with cells. It also opens up new avenues for the efficient functionalization of inorganic surfaces.

Driving h-osteoblast adhesion and proliferation on titania: peptide hydrogels decorated with growth factors and adhesive conjugates.

Hydrogels from self-assembling ionic complementary peptides have been receiving much interest from the scientific community as mimetics of the extracellular matrix that can offer three-dimensional support for cell growth or become vehicles for the delivery of stem cells or drugs. These scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In order to develop a novel bioactive titanium implant, we propose the introduction of a layer of ionic-complementary self-assembling peptides (EAbuK) on Ti whose surface has been previously sandblasted and acid etched. The peptide layer is anchored to the metal by covalent functionalization of titania with self-assembling sequences. The peptide layer has also been enriched by the insulin-like growth factor-1 incorporated to the layer and/or a conjugate obtained by chemoselective ligation between EAbuK and a sequence of 25 residues containing four GRGDSP motifs per chain. X-ray photoelectron spectroscopy studies confirmed a change in the surface composition in agreement with the proposed decorations. An evaluation of the contact angle showed a substantial change in wettability induced by the peptide layer. The human osteoblast adhesion and proliferation assays showed an increase in adhesion for the surfaces enriched with conjugate at a concentration of 3.8 × 10(-7)m and an enhanced proliferation for samples enriched with insulin-like growth factor-1 at the highest concentration tested (2.1 × 10(-5)m).

Surface-driven first-step events of nanoscale self-assembly for molecular peptide fibers: An experimental and theoretical study.

New experimental results are reported on the self-assembling behavior of EAK16-II, the first discovered ionic self-complementary peptide, incubated at ultralow concentration (10-6 M) at neutral pH onto differently charged surfaces. It is found that strongly negatively charged surfaces promote the self-assembly of flat, micrometer-long mono-molecular fibers of side-on assembled sequences, lying onto a continuous monolayer of flat-on EAK16-II molecules. These results suggest that the monomolecular EAK16-II self-assembly is driven by the peculiar matching condition between peptide and surface electrostatic properties. Molecular Mechanics simulations of the basic bimolecular interactions confirmed the experimental inferences, showing that the flat-on state is the most stable arrangement for two interacting EAK16-II sequences onto strongly negatively charged surfaces, where indeed EAK16-II ß-sheet conformation is stabilized, while the weak electrostatic interactions with mildly charged substrates promote an "entangled" EAK16-II geometry. Molecular Dynamics simulations further showed that the mobility and diffusional freedom of the peptides from the surfaces are ruled by the relative strength of peptide-surface electrostatic interactions, so that desorption probability for the peptide sequences is negligible from strongly-charged surfaces and high from mildly-charged surfaces. Furthermore, it has been found that an oligopeptide sequence lying onto two flat-on EAK16-II molecules, gains a remarkable lateral mobility, while remaining weakly bound to the surface, thus allowing the further molecular self-alignment responsible for the micrometer-long fiber formation. The reported results pave the way to the understanding and control of the subtle peptide-surface structural motifs matching enabling the formation of micrometer-long, but nanometer-wide monomolecular fibers.

Improved osteogenic differentiation of human marrow stromal cells cultured on ion-induced chemically structured poly-epsilon-caprolactone.

The ability to control cell proliferation/differentiation, using material surface, is a main goal in tissue engineering. The objective of this study was to evaluate the attachment, proliferation and differentiation to the osteoblastic phenotype of human marrow stromal cells (MSC) when seeded on poly-epsilon-caprolactone (PCL) thin films before and after irradiation with 10 keV He+. The polymeric surface was characterized as surface chemical structure and composition, roughness and morphology on the micro- and nano-scale, wettability and surface free energy parameters. MSC were obtained from patients undergoing routine hip replacement surgery, expanded in vitro and cultured on untreated PCL and He+ irradiated PCL films for up to 4-5 weeks in osteogenic medium. He+-irradiation led to slight smoothening of the surface and different nanoscale surface chemical structure, while surface free energy resulted unchanged in comparison to untreated PCL. The results from biological testing demonstrated that early attachment and further proliferation, as well as osteoblastic markers, were higher for MSC on He+-irradiated PCL. In conclusion, the change of PCL surface properties induced by ion beam irradiation is confirmed to enhance the adhesion of MSC and support their differentiation.

Ionic strength-controlled hybridization and stability of hybrids of KRAS DNA single-nucleotides: A surface plasmon resonance study.

The discrimination of a fully matched, unlabeled KRAS wild-type (WT) (C-G) target sample with respect to three of the most frequent KRAS codon mutations (G12 S (C-A), G12 R (C-C), G12C (C-T)) was investigated using an optimized detection strategy involving surface plasmon resonance (SPR), based on optimized probe-surface density and ionic strength control. The changes observed in the SPR signal were always larger for WT compared with the single-mismatch target DNA oligonucleotides, and were aligned with the theoretical energy differences between the base pair C-G, C-T, C-A, C-C. Hybridization rates of ∼106M-1s-1 were detected without the introduction of high temperature and labels, usually needed in conventional hybridization methods. One hundred percent mutation discrimination of the matched KRAS wild-type (C-G) sequence with respect to three mismatched G12C (C-T), G12 S (C-A), G12 R (C-C) target sequences was achieved.

Human bone marrow stromal cells: In vitro expansion and differentiation for bone engineering.

Stromal cells from marrow hold a great promise for bone regeneration. Even if they are already being exploited in many clinical settings, the biological basis for the source and maintenance of their proliferation/differentiation potential after in vitro isolation and expansion needs further investigation. Most studies on osteogenic differentiation of marrow stromal cells (MSC) have been performed using bone marrow from the iliac crest. In this study, MSC were derived from spare femoral bone marrow obtained during hip replacement surgery from 20 adult donors. After in vitro isolation the cells were grown in osteogenic medium, and their proliferation and differentiation analysed during in vitro expansion. We found that MSC isolated from the femur of adult patients consistently maintain an osteogenic potential. Using biochemical signals, these cells turn to fully differentiated osteoblasts with a predictable set of molecular and phenotypic events of in vitro bone deposition. When seeded on polycaprolactone-based scaffold or surfaces, the proliferation and mineralization of femur-derived MSC were modulated by the surface chemistry/topography. Despite remarkable differences between individual colony-forming ability, alkaline phosphatase production, and mineralization ability, these cells are a potential source for bone engineering, either by direct autologous reimplantation or by ex vivo expansion and reimplantation combined to a proper scaffold.

Viscoelastic properties of insoluble amphiphiles at the air/water interface.

The effects of the presence of a molecular monolayer on the dilatational properties of the air/water interface have been investigated. Two water insoluble amphiphiles, dipalmitoyl phosphatidyl choline and quercetin 3-O-palmitate, were spread onto a pendant drop and the dynamic surface pressure was measured by means of drop shape analysis. The surface dilatational elasticity and viscosity of the spread monolayers were also determined by the oscillating drop technique. Constraints on the range of measuring conditions were investigated and we demonstrated that the pressure-area isotherms derived from oscillatory dynamic measurements display phase behaviour similar to that found in equilibrium measurements, albeit at reduced resolution. Both the amphiphiles formed purely elastic films that were characterised by a dilatational modulus that depended on the surface concentration and obeyed a power scaling law. The exponent of the relationship could be related to the thermodynamic conditions prevailing at the interface. The phospholipid monolayer scaling exponent was 2.8 in a temperature range of 20-26 degrees C indicates a favourable solvency of molecules in the bidimensional matrix. A very high scaling exponent (11.8 at 7 degrees C) for quercetin palmitate was interpreted assuming that molecules self-organise in fibre-like structures. This interface structure and the phase behaviour was found consistent with observations of the surface film obtained by Brewster angle microscopy. The structured quercetin 3-O-palmitate monolayers are disrupted by temperature increase or by adding a 0.2 molar fraction of the immiscible dipalmitoyl phosphatidyl choline.

Selective protein trapping within hybrid nanowells.

Nanostructured surfaces offer a great deal in view of the control of biological processes at subcellular level. An innovative methodology has been developed to fabricate large-scale hexagonally close-packed arrays of polymer/gold nanowells of tunable diameter and depth, ranging between about 70 and 100 nm (diameter) and 15 and 40 nm (depth). Nanowell volumes down to 0.3 attolitres and nanowell densities as high as ∼10(9) wells per cm(2) could also be demonstrated. The present paper investigates the main features of protein trapping processes within the obtained nanowell arrays. Selective protein trapping, also involving orientation and biofunctionality changes, appears to be induced by the nanoconfinement. Nanomorphology measurements and antibody preferential linkages are demonstrated for human serum albumin versus lysozyme, the first being efficiently trapped within the nanocavities and the second being preferentially deposited outside them. The selective protein-dependent trapping/untrapping within the nanowells is discussed in terms of the variation in the out-diffusion coefficients of the biomolecules entering the nanowells, either as a function of the matching/mismatching of the biomolecules and nanocavity dimensions, or, alternatively, owing to the drastic conformational changes due to nanoconfinement. In this case, the trapping of large and soft human serum albumin is privileged with respect to the small and hard lysozyme. Furthermore, the observed peculiar antibody response to the confined proteins is accounted for in terms of the enhancement of their biological response following the modified accessibility of the key epitopes, which in turn suggests drastic conformational changes.

Adsorption of a cell-adhesive oligopeptide on polymer surfaces irradiated by ion beams.

The adsorption behavior of H-Arg-Gly-Asp-OH (RGD) oligopeptide on ion-irradiated polymer surfaces has been studied. The RGD-incubated surfaces of poly(ethylene terephtalate) (PET) and poly(hydroxymethylsiloxane) (PHMS) thin films, before and after irradiation with 50 keV Ar+ to 1x10(15) ions/cm2, were investigated by X-Ray Photoelectron Spectroscopy and Atomic Force Microscopy. It was found that no significant adsorption occurs on PET, while a measurable amount of RGD is preferentially adsorbed onto irradiated PHMS surfaces. The evaluated surface coverage was found to range between 5 and 12%. In situ adsorption measurements performed by using the Quartz Crystal Microbalance with Dissipation monitoring technique showed that the irradiation induced remarkable changes of mass uptake with respect to the unirradiated surfaces, mostly attributed to the change in the water adsorption capability of the irradiated surfaces. The adsorption results are discussed in terms of the ion-induced changes on the morphology, chemical structure and composition, surface free energy and surface charge.

Improved cell adhesion to ion beam-irradiated polymer surfaces.

A very strong improvement of the cell adhesion, spreading and proliferation was observed for ion beam irradiated surfaces of polyethersulphones and polyurethane. The improvement shows a characteristic ion fluence-dependence with a threshold around 1 x 10(15) ions cm-2. We have compared this improvement of surface cytocompatibility with the various ion-induced chemical and physical modifications of the polymeric surfaces, taking into account their characteristic fluence-dependence. The irradiation induced a severe compositional and chemical modification of the polymeric surfaces as detected by X-ray photoelectron spectroscopy. Contact angle measurements showed that the surface free energy was strongly modified by ion irradiation. The fluence-dependent formation of a hydrogenated amorphous carbon phase was demonstrated by Raman spectroscopy. Our findings indicate that neither the simple modification of the functional groups nor the mere elemental composition nor the modification of the surface free energy can explain the observed fluence-dependence of the cell adhesion enhancement. On the contrary, we show that this enhancement correlates with the formation of a highly specific ion-induced 'unsaturated' a-C:H phase. According to these findings, we suggest that the formation of a substantial amount of hydrogenated amorphous carbon phases is the major factor promoting the cytocompatibility of ion irradiated polymer surfaces.

Human serum albumin adsorption onto a-SiC:H and a-C:H thin films deposited by plasma enhanced chemical vapor deposition.

In the present paper, we report the study of the adsorption behavior of a model protein such as human serum albumin (HSA) onto surfaces of a-SiC:H and a-C:H thin films deposited by using the plasma-enhanced chemical vapor deposition (PECVD) technique. The surface composition and surface energy of the various substrates as well as the evaluation of the adsorbed amount of protein has been carried out by means of X-ray photoelectron spectroscopy (XPS) and contact angle measurements. It has been found that HSA tends to preferentially adsorb on Si-rich surfaces, as far as the relative amount of adsorbed HSA decreases with increasing S-C concentration. Preliminary elements of mechanistic models are proposed for the correlation between chemical factors and the observed protein adsorption behavior.

[Blood beta-carotene after oral administration of carotenoids. A dynamic test of lipid absorption]. / Beta-carotenemia da carico orale di carotinoidi. Prova dinamica di assorbimento dei lipidi.

Authors report a method on the functional investigation of intestinal epithelium, based upon the dosage of serum carotene levels after administration of carotenoids. This test takes advantage of the modality of beta-carotene absorption that it behaves like a lipid. The "beta-carotene test" has demonstrated, as regards to T.A.R.T., great sensibility and specificity over 90%. It doesn't show value overlapping between normal and coeliac children and also it shows good compliance of children and simplicity in laboratory determination. For all these characteristics the Authors suggest the utilization of "beta-carotene test" as screening tool of malabsorption.

Multiscale characterization of a chimeric biomimetic polypeptide for stem cell culture.

Mesenchymal stem cells have attracted great interest in the field of tissue engineering and regenerative medicine because of their multipotentiality and relative ease of isolation from adult tissues. The medical application of this cellular system requires the inclusion in a growth and delivery scaffold that is crucial for the clinical effectiveness of the therapy. In particular, the ideal scaffolding material should have the needed porosity and mechanical strength to allow a good integration with the surrounding tissues, but it should also assure high biocompatibility and full resorbability. For such a purpose, protein-inspired biomaterials and, in particular, elastomeric-derived polypeptides are playing a major role, in which they are expected to fulfil many of the biological and mechanical requirements. A specific chimeric protein, designed starting from elastin, resilin and collagen sequences, was characterized over different length scales. Single-molecule mechanics, aggregation properties and compatibility with human mesenchymal stem cells were tested, showing that the engineered compound is a good candidate as a stem cell scaffold to be used in tissue engineering applications.

Relationship between the fibroblastic behaviour and surface properties of RGD-immobilized PCL membranes.

In this study, poly-epsilon-caprolactone (PCL) membranes were modified with the cell adhesive peptide RGD by chemical immobilization technique. The roughness and hydrophilicity were increased after RGD immobilization and an improved cell attachment was observed.

Preliminary investigation on the balance of lead and cadmium content in milk and its by-products.

Many studies have recently been devoted to the determination of lead and cadmium in milk, in view of the toxicity of these metals and the importance of milk in the diet of infants and children. Enrichment of lead and cadmium in fatty dairy products has been sometimes noted. A systematic study on milk and its primary products (skimmed milk, cream and cheese) obtained from the same batch of milk is considered in order to define a tentative balance of these two metals. Lead and cadmium were determined by atomic-absorption spectrophotometry with graphite furnace on a solution of the ashed sample. As a consequence of the appreciable interference due to the matrix effects, the method of standard additions was adopted to determine the metals in few samples with a statistically acceptable degree of accuracy and precision. The range of the lead and cadmium content found in these products is comparable with recent literature data concerning uncontaminated samples.

Simultaneous high-performance liquid chromatographic analysis of free carotenoids and carotenoid esters.

High-performance liquid chromatography using a non-aqueous reversed phase with gradient elution on C18 columns is a powerful tool for investigating the carotenoid composition of natural samples, e.g., flower petals, and for the simultaneous detection of carotenoids of the widest possible polarity range (xanthophylls, diones, hydrocarbons and carotenoid esters). The comparison of sample extracts submitted or not to saponification allows the presence of carotenoid esters to be revealed through the appearance of the corresponding free hydroxycarotenoids. The gas chromatographic analysis of the fatty acids after alkaline hydrolysis of esters provides further confirmation. In most cases, peaks of various carotenoids were identified by comparison with standards. The wavelengths of the visible absorbance maxima of the chromatographed carotenoids as obtained on-line by the stop-flow method in the eluent system and off-line in carbon disulphide are reported. The esters appear to constitute the main carotenoid fraction in flower petals.

Chromium and nickel in roadside grapes.

Chromium and nickel concentration in roadside unwashed wine grapes and leaves is considered as a function of the distance from the suspected source, an isolated straight road. The analyses were performed with electrothermal atomization in a graphite furnace. The decrease in the concentration of the element can be described by means of a three-parameter exponential function c = A + (B - A)exp(-Cd), which allows the determination of the asymptotic level far from the emitting source (C infinity) and the pollution level (C0) at the edge of the road (d = 0). Least-squares nonlinear regression gives the estimation of the parameters A, B, and C. The results indicate that an effect is detectable up to a distance of about 50 m.

Protein adsorption and fibroblast adhesion on irradiated polysiloxane surfaces.

A very peculiar case of differential cell response towards polysiloxane surfaces of very similar composition is investigated. Poly(hydroxymethylsiloxane) (PHMS) surfaces treated either by O(2)-plasma or 6 keV Ar(+)-beams have been used to test the adhesion, proliferation and spreading of human fibroblasts. The surface chemical structure and nanomorphology were investigated by means of X-ray photoelectron spectroscopy (XPS), surface free energy measurements and atomic force microscopy (AFM). In spite of the close compositional and morphological similarity of the modified surfaces, the viability of the adhered cells, evaluated by means of optical microscopy and epifluorescence microscopy, was found to be very different in the two cases. The study of the features of the adsorbed protein adlayer on the two types of surfaces was performed by XPS and AFM and indicated that the overall cell behavior is connected to a quite different protein aggregation process, occurring respectively on the plasma- and Ar(+)-modified polysiloxane surfaces. It is suggested that the specific biological response of the modified surfaces is determined by the chemical structure at the nanometric level.