Nanoroughness, Surface Chemistry and Drug Delivery Control by Atmospheric Plasma Jet on Implantable Devices

Implantable devices need specific tailored surface morphologies and chemistries to interact with the living systems or to actively induce a biological response also by the release of drugs or proteins. These customised requirements foster technologies that can be implemented in additive manufacturing systems. Here we present a novel approach based on spraying processes that allows to control separately topographic features in the submicron range ( 3d 60 nm - 2 \ub5m), ammine or carboxylic chemistry and fluorophore release even on temperature sensitive biodegradable polymers such as polycaprolactone (PCL). We developed a two-steps process with a first deposition of 220 nm silica and poly(lactic-co-glycolide) (PLGA) fluorescent nanoparticles by aerosol followed by the deposition of a fixing layer by atmospheric pressure plasma jet (APPJ). The nanoparticles can be used to create the nano-roughness and to include active molecule release, while the capping layer ensures stability and the chemical functionalities. The process is enabled by a novel APPJ which allows deposition rates of 10 - 20 nm\ub7s-1 at temperatures lower than 50 \ub0C using argon as process gas. This approach was assessed on titanium alloys for dental implants and on PCL films. The surfaces were characterized by FT-IR, AFM and SEM. Titanium alloys were tested with pre-osteoblasts murine cells line, while PCL film with fibroblasts. Cell behaviour was evaluated by viability and adhesion assays, protein adsorption, cell proliferation, focal adhesion formation and SEM. The release of a fluorophore molecule was assessed in the cell growing media, simulating a drug release. Osteoblast adhesion on the plasma treated materials increased by 20% with respect to commercial titanium alloys implants. Fibroblast adhesion increased by a 100% compared to smooth PCL substrate. The release of the fluorophore by the dissolution of the PLGA nanoparticles was verified and the integrity of the encapsulated drug model confirmed

Physical–chemical properties of biogenic selenium nanostructures produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite (www.frontiersin.org) was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by www.frontiersin.org upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes

Electrochemical Immunosensor for Detection of IgY in Food and Food Supplements

Immunoglobulin Y is a water-soluble protein present in high concentration in hen serum and egg yolk. IgY has applications in many fields, e.g., from food stuff to the mass production of antibodies. In this work, we have implemented an electrochemical immunosensor for IgY based on templated nanoelectrodes ensembles. IgY is captured by the templating polycarbonate and reacted with anti-IgY labeled with horseradish peroxidase. In the presence of H2O2 and methylene blue as the redox mediator, an electrocatalytic current is generated which scales with IgY concentration in the sample. After optimizing the extracting procedure, the immunosensor was applied for analysis of fresh eggs and food integrators. The data obtained with the biosensor were validated by SDS-PAGE and Western blot measurements

Physical–Chemical Properties of Biogenic Selenium Nanostructures Produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite () was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes

Structural changes in the BH3 domain of SOUL protein upon interaction with the anti-apoptotic protein Bcl-xL

The SOUL protein is known to induce apoptosis by provoking the mitochondrial permeability transition, and a sequence homologous with the BH3 (Bcl-2 homology 3) domains has recently been identified in the protein, thus making it a potential new member of the BH3-only protein family. In the present study, we provide NMR, SPR (surface plasmon resonance) and crystallographic evidence that a peptide spanning residues 147–172 in SOUL interacts with the anti-apoptotic protein Bcl-xL. We have crystallized SOUL alone and the complex of its BH3 domain peptide with Bcl-xL, and solved their three-dimensional structures. The SOUL monomer is a single domain organized as a distorted β-barrel with eight anti-parallel strands and two α-helices. The BH3 domain extends across 15 residues at the end of the second helix and eight amino acids in the chain following it. There are important structural differences in the BH3 domain in the intact SOUL molecule and the same sequence bound to Bcl-xL

Expression, purification and structural characterization of three human proteins: apolipoprotein M, heme-binding protein 2 and folate receptor \u3b1

In questo lavoro di tesi ci si \ue8 occupati dell\u2019espressione, della purificazione e della cristallizzazione di tre proteine umane (l\u2019apolipoproteina M, il recettore del folato \u3b1 e la proteina SOUL) con lo scopo finale di determinarne la struttura tridimensionale mediante analisi di diffrazione di raggi X. L\u2019apolipoproteina M umana \ue8 stata espressa utilizzando il lievito metilotrofico P. pastoris. La proteina ricombinante cos\uec ottenuta \ue8 stata purificata tramite cromatografia a scambio ionico, isoelettrofocalizzazione preparativa, gel filtrazione e cromatografia ad interazione idrofobica. Per ottenere una proteina pi\uf9 omogenea \ue8 stato espresso e purificato anche il mutante Asn135Gln, privo del sito di glicosilazione. Le prove di cristallizzazione hanno dato esito positivo con la proteina mutata, anche se i cristalli fino ad ora ottenuti non sono idonei per gli esperimenti di diffrazione di raggi X. L\u2019espressione eterologa del recettore del folato umano ha dato parecchi problemi, nonostante siano stati provati diversi sistemi di espressione (P. pastoris, baculovirus e N. benthamiana). Solo una piccola quantit\ue0 di proteina ricombinante \ue8 stata ottenuta (da P. pastoris) e purificata (mediante cromatografia a scambio ionico e gel filtrazione). Nessuna delle condizioni di cristallizzazione testata ha avuto successo, probabilmente a causa della bassa concentrazione della proteina utilizzata in tali prove. La proteina SOUL (heme-binding protein 2) \ue8 stata espressa in E. coli e purificata tramite cromatografia di affinit\ue0, sfruttando la coda di sei istidine aggiunta all\u2019estremit\ue0 C-terminale della proteina. La SOUL ricombinante \ue8 stata cristallizzata sia come apopoteina sia come oloproteina (complesso SOUL/emina). Gli studi preliminari di diffrazione di raggi X mostrano la presenza di sei molecole nella cella unitaria. Non \ue8 stata inoltre rilevata alcuna significativa differenza tra la forma apo- e la forma olo-. Ulteriori studi suggeriscono che l\u2019emina non sia legata alla proteina, poich\ue9 il picco corrispondente al ferro non \ue8 stato trovato nello spettro di fluorescenza ai raggi X ottenuto dai cristalli. Al momento sono in corso i tentativi di risolverne la struttura tridimensionale per mezzo di sostituzione isomorfa multipla, multiwavelength anomalous diffraction e sostituzione molecolare.This thesis work was aimed at the expression, purification and crystallization of three human proteins (apolipoprotein M, folate receptor \u3b1 and SOUL protein) in order to determine their three-dimensional structure by means of X-ray protein crystallography. Human apolipoprotein M was expressed using the methylotrophic yeast P. pastoris. The recombinant protein was purified by ion-exchange chromatography, preparative isoelectric focusing, gel filtration, and Lipidex-1000 chromatography. In order to obtain a more homogeneous protein, the non-glycosylated mutant (Asn135Gln) was also expressed and purified. The crystallization trials gave some positive results with mutated apoM, although the crystals are still not suitable for X-ray diffraction experiments. The heterologous expression of the human FR-a was troublesome, and although different expression systems (P. pastoris, baculovirus, and N. benthamiana) were tested, only a low amount of recombinant protein was obtained (from P. pastoris) and purified (by ion-exchange chromatography and gel filtration). However non of the crystallization conditions tested was successful, probably due to the low protein concentration. Human SOUL (heme-binding protein 2) was expressed in E. coli and purified by immobilized metal ion affinity chromatography, using the hexa-histidine tag added to the C-terminus of the protein. The recombinant SOUL was crystallized both as apoprotein and as a complex in the presence of hemin. The preliminary X-ray diffraction analysis shows the presence of six molecules in the unit cell, and no significant differences between the apoand the holoprotein were found. Further studies suggest that hemin is not bound to the protein, since the Fe peak could not be found in the X-ray fluorescence spectrum of the crystals. Attempts to solve the three-dimensional structure by means of multiple isomorphous replacement, multiwavelength anomalous diffraction and molecular replacement are still in progress

Functionalization of Mesoporous Silica Nanoparticles with Organosilanes: Experimental Evidence of the Interaction Between Organic Groups and Silica Surface

The functionalization of mesoporous silica nanoparticles (MSNs) is a very important step in the preparation of these systems for a variety of applications. The surface of MSNs can be widely functionalized with different organic groups. There are essentially two ways to covalently modify the surface of nanoparticles: co-condensation and post-grafting. Generally, for both of these methods, the precursors are: [(R’O)3SiR] and [Cl3SiR]. The paper summarizes the main experimental contributions and the recent advances in the study of interactions among organosilanes and the MSNs surface. In particular, it provides relevant and innovative examples of Solid State and Solution NMR (SSNMR and SolNMR) and Fourier Transform InfraRed (FTIR) spectroscopy that highlight different possible approaches to understand the MSNs-pendants interaction

Light Conversion upon Photoexcitation of NaBiF₄:Yb³⁺/Ho³⁺/Ce³⁺ Nanocrystalline Particles

NaBiF4 nanocrystalline particles were synthesized by means of a facile precipitation synthesis route to explore upconversion emission properties when doped with lanthanide ions. In particular, the incorporation of the Yb3+-Ho3+-Ce3+ triad with controlled ion concentration facilitates near-IR pumping conversion into visible light, with the possibility of color emission tuning depending on Ce3+ doping amount. We observed that introducing a Ce3+ content up to 20 at.% in NaBiF4:Yb3+/Ho3+, the chromaticity progressively turns from green for the Ce3+ undoped system to red. This is due to cross-relaxation mechanisms between Ho3+ and Ce3+ ions that influence the relative efficiency of the overall upconversion pathways, as discussed on the basis of a theoretical rate equation model. Furthermore, experimental results suggest that the photoexcitation of intra-4f Ho3+ transitions with light near the UV-visible edge can promote downconverted Yb3+ near-IR emission through quantum cutting triggered by Ho3+-Yb3+ energy transfer mechanisms. The present study evidences the potentiality of the developed NaBiF4 particles for applications that exploit lanthanide-based light frequency conversion and multicolor emission tuning

Light Conversion upon Photoexcitation of NaBiF4:Yb3+/Ho3+/Ce3+ Nanocrystalline Particles

NaBiF4 nanocrystalline particles were synthesized by means of a facile precipitation synthesis route to explore upconversion emission properties when doped with lanthanide ions. In particular, the incorporation of the Yb3+-Ho3+-Ce3+ triad with controlled ion concentration facilitates near-IR pumping conversion into visible light, with the possibility of color emission tuning depending on Ce3+ doping amount. We observed that introducing a Ce3+ content up to 20 at.% in NaBiF4:Yb3+/Ho3+, the chromaticity progressively turns from green for the Ce3+ undoped system to red. This is due to cross-relaxation mechanisms between Ho3+ and Ce3+ ions that influence the relative efficiency of the overall upconversion pathways, as discussed on the basis of a theoretical rate equation model. Furthermore, experimental results suggest that the photoexcitation of intra-4f Ho3+ transitions with light near the UV-visible edge can promote downconverted Yb3+ near-IR emission through quantum cutting triggered by Ho3+-Yb3+ energy transfer mechanisms. The present study evidences the potentiality of the developed NaBiF4 particles for applications that exploit lanthanide-based light frequency conversion and multicolor emission tuning

Vibrational and structural investigation of SOUL protein single crystals by using micro-Raman spectroscopy

Protein SOUL is a new member of the recently discovered putative heme-binding protein family called SOUL/HEBP and, to date, no structural information exists for this protein. Here, micro-Raman spectroscopy is used to study the vibrational properties of single crystals obtained from recombinant protein SOUL by means of two different optimization routes. This spectroscopic approach offers the valuable advantage of the in-situ collection of experimental data from protein crystals, placed onto a hanging-drop plate, under the same conditions used to grow the crystals. By focusing on the regions of amides I and III bands, some secondary structure characteristic features have been recognized. Moreover, some sidechain marker bands were observed in the Raman spectra of SOUL crystals and the unambiguous assignment of these peaks inferred by comparing the experimental Raman spectra of pure amino acids and their Raman intensities computed using quantum chemical calculations. Our comparative analysis allows to get a deeper understanding of the side-chain environments and of the interactions involving these specific amino acids in the two different SOUL crystals