Dispersion and Functionalization of Nanoparticles Synthesized by Gas Aggregation Source: Opening New Routes Toward the Fabrication of Nanoparticles for Biomedicine.

The need to find new nanoparticles for biomedical applications is pushing the limits of the fabrication methods. New techniques with versatilities beyond the extended chemical routes can provide new insight in the field. In particular, gas aggregation sources offer the possibility to fabricate nanoparticles with controlled size, composition, and structure out of thermodynamics. In this context, the milestone is the optimization of the dispersion and functionalization processes of nanoparticles once fabricated by these routes as they are generated in the gas phase and deposited on substrates in vacuum or ultra-high vacuum conditions. In the present work we propose a fabrication route in ultra-high vacuum that is compatible with the subsequent dispersion and functionalization of nanoparticles in aqueous media and, which is more remarkable, in one single step. In particular, we will present the fabrication of nanoparticles with a sputter gas aggregation source using a Fe50B50 target and their further dispersion and functionalization with polyethyleneglycol (PEG). Characterization of these nanoparticles is carried out before and after PEG functionalization. During functionalization, significant boron dissolution occurs, which facilitates nanoparticle dispersion in the aqueous solution. The use of different complementary techniques allows us to prove the PEG attachment onto the surface of the nanoparticles, creating a shell to make them biocompatible. The result is the formation of nanoparticles with a structure mainly composed by a metallic Fe core and an iron oxide shell, surrounded by a second PEG shell dispersed in aqueous solution. Relaxivity measurements of these PEG-functionalized nanoparticles assessed their effectiveness as contrast agents for magnetic resonance imaging (MRI) analysis. Therefore, this new fabrication route is a reliable alternative for the synthesis of nanoparticles for biomedicine.

FTIR and XPS studies of protein adsorption onto functionalized bioactive glass.

Adsorption and structural changes that occur upon interaction between methemoglobin (MetHb) and 5-methyl-aminomethyl-uridine forming enzyme (MnmE) with the surface of a bioactive glass (BG) were investigated by Fourier Transform Infrared (FTIR) spectroscopy and X-ray Photoelectron Spectroscopy (XPS). The effect of glutaraldehyde (GA) as a coupling agent for protein adsorption on BG was also investigated. The comparative analysis of FTIR spectra recorded from lyophilized proteins and from bioactive glass surface after protein adsorption was considered in order to obtain information about the changes in the secondary structure of the proteins. XPS data were used to determine the surface coverage. The unfolding of adsorbed proteins due to interactions between the internal hydrophobic protein domains and the hydrophobic BG surface was evidenced. After adsorption, the amount of α-helix decreases and less ordered structures (turns, random coils and aggregates) are preponderant. These changes are less pronounced on the BG functionalized with GA, suggesting that the treatment with GA preserves significantly larger amounts of α-helices in the structure of both proteins after adsorption.

Structure and dynamics of spin-labeled insulin entrapped in a silica matrix by the sol-gel method.

The structure and conformational dynamics of insulin entrapped into a silica matrix was monitored during the sol to maturated-gel transition by electron paramagnetic resonance (EPR) spectroscopy. Insulin was successfully spin-labeled with iodoacetamide and the bifunctional nitroxide reagent HO-1944. Room temperature continuous wave (cw) EPR spectra of insulin were recorded to assess the mobility of the attached spin labels. Insulin conformation and its distribution within the silica matrix were studied using double electron-electron resonance (DEER) and low-temperature cw-EPR. A porous oxide matrix seems to form around insulin molecules with pore diameters in the order of a few nanometers. Secondary structure of the encapsulated insulin investigated by Fourier transform infrared spectroscopy proved a high structural integrity of insulin even in the dried silica matrix. The results show that silica encapsulation can be used as a powerful tool to effectively isolate and functionally preserve biomolecules during preparation, storage, and release.

Attachment and conformational changes of collagen on bioactive glass surface.

The proteins adsorption on biomaterials surface leads to changes in their structural conformation that may further influence the adhesion, migration and growth of cells. The aim of this study was to examine the attachment of collagen (calf skin type I) on bioactive glass powders and the conformational changes of the protein. Scanning electron microscopy analysis and X-ray photoelectron spectroscopy measurements indicate that the collagen cover the glass surface in a nanometric thin layer. The infrared amide I absorption signal shows pronounced changes in the secondary structure of the adsorbed collagen.

The attachment affinity of hemoglobin toward silver-containing bioactive glass functionalized with glutaraldehyde.

Bioactive glasses belonging to the 56SiO2·(40 - x)CaO·4P2O5·xAg2O system, with x = 0, 2, and 8 mol %, were surface functionalized with the protein coupling agent glutaraldehyde (GA) and further evaluated in terms of hemoglobin affinity. The bare and GA-functionalized samples were investigated before and after protein attachment, by electron paramagnetic resonance (EPR) spectroscopy combined with spin-labeling procedure. Methanethiosulfonate spin label was used to explore the local environment of ß-93 cysteine in horse hemoglobin, in terms of spin label side chain mobility. The EPR simulation methods were employed to quantify the rotational correlational times and fraction of the immobilized spin labels. The EPR absorption spectrum was further exploited to estimate the amount of hemoglobin loaded on the substrates. The surface elemental composition obtained by X-ray photoelectron spectroscopy revealed similar tendency in terms of surface coverage. Changes in surface architecture, that is, changes in surface morphology after protein coverage, were observed by scanning electron microscopy. It was concluded that GA improves the stability of protein attachment and induces polymerization of hemoglobin molecules.

Bioactivity and protein attachment onto bioactive glasses containing silver nanoparticles.

There is much interest in silver containing glasses for use in bone replacement owing to the demonstrated antibacterial effect. In this work, 2 and 8 mol % of silver was added during the sol-gel process to the composition of a bioactive glass belonging to CaO-SiO(2 -P(2)O(5) system. The samples were characterized by means of ultraviolet-visible spectroscopy and X-ray photoelectron spectroscopy (XPS) techniques to demonstrate that the silver is embedded into the glass matrix as nanoparticles. Bioactivity test in simulated body fluid proved that the presence of silver in the bioactive glass composition, even in high amount, preserve or even improve the bioactivity of the starting glass, and consequently, leads to the carbonated apatite formation, which is the prerequisite for bioactive materials to bond with living bones. Complementary information proving these findings were delivered by performing X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy, and XPS measurements. The presence of silver also improves protein binding capability to the bioactive glass surface as demonstrated by cw-electron paramagnetic resonance experiments and XPS measurements.