Scaling Up Magnetic Nanobead Synthesis with Improved Stability for Biomedical Applications.

The growing interest in multifunctional nano-objects based on polymers and magnetic nanoparticles for biomedical applications motivated us to develop a scale-up protocol to increase the yield of polymeric magnetic nanobeads while aiming at keeping the structural features at optimal conditions. The protocol was applied to two different types of magnetic ferrite nanoparticles: the Mn-ferrite selected for their properties as contrast agents in magnetic resonance imaging and iron oxide nanostar shaped nanoparticles chosen for their heat performance in magnetic hyperthermia. At the same time, some experiments on surface functionalization of nanobeads with amino modified polyethyelene glycol (PEG) molecules have provided further insight into the formation mechanism of magnetic nanobeads and the need to cross-link the polymer shell to improve the stability of the beads, making them more suitable for further manipulation and use. The present work summarizes the most important parameters required to be controlled for the upscaling of nanobead synthesis in a bench protocol and proposes an alternative cross-linking strategy based on prefunctionalization of the polymer prior to the nanobead formation as a key parameter to improve the nanobead structural stability in solutions at different pHs and during surface functionalization.

Laser-Fabricated Fluorescent, Ligand-Free Silicon Nanoparticles: Scale-up, Biosafety, and 3D Live Imaging of Zebrafish under Development.

This work rationalizes the scalable synthesis of ultrasmall, ligand-free silicon nanomaterials via liquid-phase pulsed laser ablation process using picosecond pulses at ultraviolet wavelengths. Results showed that the irradiation time drives hydrodynamic NP size. Isolated, monodisperse Si-NPs are obtained at high yield (72%) using post-treatment process. The obtained Si-NPs have an average size of ∼10 nm (not aggregated) and display photoemission in the green spectral range. We directly characterized the ligand-free Si-NPs in a vertebrate animal (zebrafish) and assessed their toxicity during the development. In vivo assay revealed that Si-NPs are found inside in all the early life stages of embryos and larvae growth, showing that the biosafety of Si-NPs and malformation types are independent of the Si-NP dose. Si-NPs were directly imaged inside developing embryos by spinning disk-imaging technique with optical sectioning capability. We showed that Si-NPs can passively enter inside embryos by the pore canals of chorion, can diffuse in the circulatory system, i.e., blood vessel, and accumulate inside larvae midgut and yolk sac, and in the eye lens, indicating the crossing of the blood barrier.

Multilayered Magnetic Nanobeads for the Delivery of Peptides Molecules Triggered by Intracellular Proteases.

In this work, the versatility of layer-by-layer technology was combined with the magnetic response of iron oxide nanobeads to prepare magnetic mesostructures with a degradable multilayer shell into which a dye quenched ovalbumin conjugate (DQ-OVA) was loaded. The system was specifically designed to prove the protease sensitivity of the hybrid mesoscale system and the easy detection of the ovalbumin released. The uptake of the nanostructures in the breast cancer cells was followed by the effective release of DQ-OVA upon activation via the intracellular proteases degradation of the polymer shells. Monitoring the fluorescence rising due to DQ-OVA digestion and the cellular dye distribution, together with the electron microscopy studying, enabled us to track the shell degradation and the endosomal uptake pathway that resulted in the release of the digested fragments of DQ ovalbumin in the cytosol.

Facile fabrication of bioactive ultra-small protein-hydroxyapatite nanoconjugates via liquid-phase laser ablation and their enhanced osteogenic differentiation activity.

Hydroxyapatite bioactive complexes are being increasingly recognized as effective available means in regenerative medicine. Conventional technologies for their synthesis have drawbacks from a synthetic standpoint, mainly requiring high temperatures and multi-step processes. Here, we show that ultra-small hydroxyapatite conjugated-nanoparticles (Ha-CNPs) can be obtained at room temperature by Pulsed Laser Ablation (PLA) directly in protein solution using picosecond pulses at near infrared wavelengths. The results showed that the nanoparticle size was driven by the concentration of the protein. Using this approach, we obtained aqueous soluble and ultra-small crystalline nanoparticles of ≈3 nm diameter coated with protein molecules (surface coverage ≈ 5.5 pmol cm-2; zeta potential ≈-33.5 mV). These nanoparticles showed low cytotoxicity in vitro compared to chemically synthesized nanoparticles, and revealed proliferative and osteoinductive effects on human bone marrow mesenchymal stem cells (hMSCs). The resulting enhanced cell osteogenic differentiation suggested that our PLA-based synthetic approach might be exploited in novel applications of regenerative medicine.

Biocompatibility and biodistribution of functionalized carbon nano-onions (f-CNOs) in a vertebrate model.

Functionalized carbon nano-onions (f-CNOs) are of great interest as platforms for imaging, diagnostic and therapeutic applications due to their high cellular uptake and low cytotoxicity. To date, the toxicological effects of f-CNOs on vertebrates have not been reported. In this study, the possible biological impact of f-CNOs on zebrafish during development is investigated, evaluating different toxicity end-points such as the survival rate, hatching rate, and heart beat rate. Furthermore, a bio-distribution study of boron dipyrromethene (BODIPY) functionalized CNOs in zebrafish larvae is performed by utilizing inverted selective plane illumination microscopy (iSPIM), due to its intrinsic capability of allowing for fast 3D imaging. Our in vivo findings indicate that f-CNOs exhibit no toxicity, good biocompatibility (in the concentration range of 5-100 µg mL-1) and a homogenous biodistribution in zebrafish larvae.

Direct surface modification of ligand-free silicon quantum dots prepared by femtosecond laser ablation in deionized water.

Amine-terminated, ultra-small silicon nanoparticles (Si-NPs) were prepared in one step avoiding the conventional chemical or thermal treatment of Si surface, by introducing organosilane in Si-NPs colloidal solution freshly prepared by ultra-fast laser ablation of silicon target in deionized water. Surface chemistry studies of Si-NPs conducted by Raman and Fourier infrared spectroscopy demonstrated the hydroxyl-terminated surface of Si-NPs. The reactivity of hydroxyl-terminated surface with aminopropyltriethoxysilane in aqueous solution was investigated. Electron microscopy, dynamic light scattering, infrared spectroscopy and stability studies confirmed the successful functionalization of Si-NPs leading to 5nm Si dots covered by aminopropyltriethoxysilane thick layer. Detailed infrared spectroscopy analysis of the Si-O-Si region as a function of immersion time revealed the formation of interfacial Si-O bonds between the organosilane and hydroxyl groups of the nanoparticles. The biocompatible Si nanostructure containing amine functional group prepared using a one-step green protocol opens the route for biomedical applications and successful translation into clinical setting, as bio-labels, contrast agents and vector delivery.

Extensive Characterization of Oxide-Coated Colloidal Gold Nanoparticles Synthesized by Laser Ablation in Liquid.

Colloidal gold nanoparticles are a widespread nanomaterial with many potential applications, but their aggregation in suspension is a critical issue which is usually prevented by organic surfactants. This solution has some drawbacks, such as material contamination and modifications of its functional properties. The gold nanoparticles presented in this work have been synthesized by ultra-fast laser ablation in liquid, which addresses the above issues by overcoating the metal nanoparticles with an oxide layer. The main focus of the work is in the characterization of the oxidized gold nanoparticles, which were made first in solution by means of dynamic light scattering and optical spectroscopy, and then in dried form by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and finally by surface potential measurements with atomic force microscopy. The light scattering assessed the nanoscale size of the formed particles and provided insight in their stability. The nanoparticles' size was confirmed by direct imaging in transmission electron microscopy, and their crystalline nature was disclosed by X-ray diffraction. The X-ray photoelectron spectroscopy showed measurements compatible with the presence of surface oxide, which was confirmed by the surface potential measurements, which are the novel point of the present work. In conclusion, the method of laser ablation in liquid for the synthesis of gold nanoparticles has been presented, and the advantage of this physical approach, consisting of coating the nanoparticles in situ with gold oxide which provides the required morphological and chemical stability without organic surfactants, has been confirmed by using scanning Kelvin probe microscopy for the first time.

Fabrication of hybrid nanocomposite scaffolds by incorporating ligand-free hydroxyapatite nanoparticles into biodegradable polymer scaffolds and release studies.

We report on the optical fabrication approach of preparing free-standing composite thin films of hydroxyapatite (HA) and biodegradable polymers by combining pulsed laser ablation in liquid and mask-projection excimer laser stereolithography (MPExSL). Ligand-free HA nanoparticles were prepared by ultrafast laser ablation of a HA target in a solvent, and then the nanoparticles were dispersed into the liquid polymer resin prior to the photocuring process using MPExSL. The resin is poly(propylene fumarate) (PPF), a photo-polymerizable, biodegradable material. The polymer is blended with diethyl fumarate in 7:3 w/w to adjust the resin viscosity. The evaluation of the structural and mechanical properties of the fabricated hybrid thin film was performed by means of SEM and nanoindentation, respectively, while the chemical and degradation studies were conducted through thermogravimetric analysis, and FTIR. The photocuring efficiency was found to be dependent on the nanoparticle concentration. The MPExSL process yielded PPF thin films with a stable and homogenous dispersion of the embedded HA nanoparticles. Here, it was not possible to tune the stiffness and hardness of the scaffolds by varying the laser parameters, although this was observed for regular PPF scaffolds. Finally, the gradual release of the hydroxyapatite nanoparticles over thin film biodegradation is reported.

Continuous thermal collapse of the intrinsically disordered protein tau is driven by its entropic flexible domain.

The tau protein belongs to the category of Intrinsically Disordered Proteins (IDP), which in their native state lack a folded structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilizing the microtubules' (MTs) surfaces in the axons of the neurons. Tau is mainly composed by two domains: (i) the binding domain that tightly bounds the MT surfaces and (ii) the projection domain that exerts a long-range entropic repulsive force and thus provides the proper spacing between adjacent MTs. Tau is also involved in the genesis and in the development of the Alzheimer disease when it detaches from MT surfaces and aggregates in paired helical filaments. Unfortunately, the molecular mechanisms behind these phenomena are still unclear. Temperature variation, rarely considered in biological studies, is here used to provide structural information on tau correlated to its role as an entropic spacer between adjacent MTs surfaces. In this paper, by means of small-angle X-ray scattering and molecular dynamics simulation, we demonstrate that tau undergoes a counterintuitive collapse phenomenon with increasing temperature. A detailed analysis of our results, performed by the Ensemble Optimization Method, shows that the thermal collapse is coupled to the occurrence of a transient long-range contact between a region encompassing the end of the proline-rich domain P2 and the first part of the repeats domain, and the region of the N-terminal domain entailing residues 80-150. Interestingly these two regions involved in the tau temperature collapse belong to the flexible projection domain that acts as an entropic bristle and regulates the MTs' architecture. Our results show that temperature is an important parameter that influences the dynamics of the tau projection domain, and hence its entropic behavior.