Assessment of laser-synthesized Si nanoparticle effects on myoblast motility, proliferation and differentiation: towards potential tissue engineering applications.

Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 µg mL-1. Based on these findings, three concentrations of 14, 28 and 56 µg mL-1 were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 µg mL-1, the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 µg mL-1. Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine.

Chitosan-coated ultrapure silicon nanoparticles produced by laser ablation: biomedical potential in nano-oncology as a tumor-targeting nanosystem.

Ultrapure silicon nanoparticles (SiNPs) produced by femtosecond laser ablation in water have attracted great interest in the area of cancer therapy as they are efficient as photosensitizers in photodynamic therapy modality and can induce cell hyperthermia under radiofrequency radiation. Recently, we showed that these biocompatible nanoparticles were not able to reach tumors after intravenous injection in mice due to their rapid clearance from the bloodstream. In order to increase their half-life time and therefore their chances to reach and accumulate in tumors by an enhanced permeation retention (EPR) effect, a capping agent on SiNP surface acting as a colloidal stabilizer suspension is required. In this regard, this work focuses for the first time on the functionalization of SiNPs through the modification of their surface by chitosan (SiNPs-CH) in order to enhance their therapeutic properties in cancer therapy. Here, in vivo experiments were carried out during 15 days on nude mice developing a subcutaneously grafted malignant human brain tumor (glioblastoma). The characterization of SiNPs-CH showed an average hydrodynamic size of around 142 ± 65 nm as well as a relatively neutral charge (-5.2 mV) leading to a high colloidal suspension stability. The point of our work concerns the improvement of the biodistribution of SiNPs-CH with regard to tumors, the bloodstream, and organs. After the intravenous administration of 20 mg kg-1, all the studied parameters (animal behavior, organs' morphology, and histopathology) were in accord with the absence of toxicity due to SiNPs-CH, confirming their biocompatibility and even size and surface charge were modified compared to bare nanoparticles. Moreover an increased time in the bloodstream circulation of up to 7 days was observed, indicating the stealth of the nanoparticles, which could escape opsonization and premature elimination by macrophages and the reticuloendothelial system. As evidenced by silicon assessment, the interaction of the SiNPs-CH with the liver and spleen was significantly reduced compared to the bare nanoparticles. At the same time, SiNPs-CH were concentrated progressively in tumors from 12.03% after 1 day up to 39.55% after 7 days, confirming their uptake by the tumor microenvironment through the enhanced permeability retention effect. Subsequently, the silicon level declined progressively down to 33.6% after 15 days, evidencing the degradation of pH-sensitive SiNPs-CH under the acidic tumor microenvironment. Taken together, the stealthy SiNPs-CH exhibited an ideal biodistribution profile within the tumor microenvironment with a sustainable biodegradation and elimination profile, indicating their promising application in the nano-oncology field as a tumor-targeting system.

Laser-Ablative Synthesis of Ultrapure Magneto-Plasmonic Core-Satellite Nanocomposites for Biomedical Applications.

The combination of magnetic and plasmonic properties at the nanoscale promises the development of novel synergetic image-guided therapy strategies for the treatment of cancer and other diseases, but the fabrication of non-contaminated magneto-plasmonic nanocomposites suitable for biological applications is difficult within traditional chemical methods. Here, we describe a methodology based on laser ablation from Fe target in the presence of preliminarily ablated water-dispersed Au nanoparticles (NPs) to synthesize ultrapure bare (ligand-free) core-satellite nanostructures, consisting of large (several tens of nm) Fe-based core decorated by small (mean size 7.5 nm) Au NPs. The presence of the Fe-based core conditions a relatively strong magnetic response of the nanostructures (magnetization of >12.6 emu/g), while the Au NPs-based satellite shell provides a broad extinction peak centered at 550 nm with a long tale in the near-infrared to overlap with the region of relative tissue transparency (650-950 nm). We also discuss possible mechanisms responsible for the formation of the magnetic-plasmonic nanocomposites. We finally demonstrate a protocol to enhance colloidal stability of the core-satellites in biological environment by their coating with different polymers. Exempt of toxic impurities and combining strong magnetic and plasmonic responses, the formed core-satellite nanocomposites can be used in biomedical applications, including photo- and magneto-induced therapies, magnetic resonance imaging or photoacoustic imaging.

Short-Pulse Lasers: A Versatile Tool in Creating Novel Nano-/Micro-Structures and Compositional Analysis for Healthcare and Wellbeing Challenges.

Driven by flexibility, precision, repeatability and eco-friendliness, laser-based technologies have attracted great interest to engineer or to analyze materials in various fields including energy, environment, biology and medicine. A major advantage of laser processing relies on the ability to directly structure matter at different scales and to prepare novel materials with unique physical and chemical properties. It is also a contact-free approach that makes it possible to work in inert or reactive liquid or gaseous environment. This leads today to a unique opportunity for designing, fabricating and even analyzing novel complex bio-systems. To illustrate this potential, in this paper, we gather our recent research on four types of laser-based methods relevant for nano-/micro-scale applications. First, we present and discuss pulsed laser ablation in liquid, exploited today for synthetizing ultraclean "bare" nanoparticles attractive for medicine and tissue engineering applications. Second, we discuss robust methods for rapid surface and bulk machining (subtractive manufacturing) at different scales by laser ablation. Among them, the microsphere-assisted laser surface engineering is detailed for its appropriateness to design structured substrates with hierarchically periodic patterns at nano-/micro-scale without chemical treatments. Third, we address the laser-induced forward transfer, a technology based on direct laser printing, to transfer and assemble a multitude of materials (additive structuring), including biological moiety without alteration of functionality. Finally, the fourth method is about chemical analysis: we present the potential of laser-induced breakdown spectroscopy, providing a unique tool for contact-free and space-resolved elemental analysis of organic materials. Overall, we present and discuss the prospect and complementarity of emerging reliable laser technologies, to address challenges in materials' preparation relevant for the development of innovative multi-scale and multi-material platforms for bio-applications.

Smart Electrospun Hybrid Nanofibers Functionalized with Ligand-Free Titanium Nitride (TiN) Nanoparticles for Tissue Engineering.

Herein, we report the fabrication and characterization of novel polycaprolactone (PCL)-based nanofibers functionalized with bare (ligand-free) titanium nitride (TiN) nanoparticles (NPs) for tissue engineering applications. Nanofibers were prepared by a newly developed protocol based on the electrospinning of PCL solutions together with TiN NPs synthesized by femtosecond laser ablation in acetone. The generated hybrid nanofibers were characterised using spectroscopy, microscopy, and thermal analysis techniques. As shown by scanning electron microscopy measurements, the fabricated electrospun nanofibers had uniform morphology, while their diameter varied between 0.403 ± 0.230 µm and 1.1 ± 0.15 µm by optimising electrospinning solutions and parameters. Thermal analysis measurements demonstrated that the inclusion of TiN NPs in nanofibers led to slight variation in mass degradation initiation and phase change behaviour (Tm). In vitro viability tests using the incubation of 3T3 fibroblast cells in a nanofiber-based matrix did not reveal any adverse effects, confirming the biocompatibility of hybrid nanofiber structures. The generated hybrid nanofibers functionalized with plasmonic TiN NPs are promising for the development of smart scaffold for tissue engineering platforms and open up new avenues for theranostic applications.

Laser Ablation-Assisted Synthesis of Plasmonic Si@Au Core-Satellite Nanocomposites for Biomedical Applications.

Owing to strong plasmonic absorption and excellent biocompatibility, gold nanostructures are among best candidates for photoacoustic bioimaging and photothermal therapy, but such applications require ultrapure Au-based nanoformulations of complex geometry (core-shells, nanorods) in order to shift the absorption band toward the region of relative tissue transparency (650-1000 nm). Here, we present a methodology for the fabrication of Si@Au core-satellite nanostructures, comprising of a Si core covered with small Au nanoparticles (NP), based on laser ablative synthesis of Si and Au NPs in water/ethanol solutions, followed by a chemical modification of the Si NPs by 3-aminopropyltrimethoxysilane (APTMS) and their subsequent decoration by the Au NPs. We show that the formed core-satellites have a red-shifted plasmonic absorption feature compared to that of pure Au NPs (520 nm), with the position of the peak depending on APTMS amount, water-ethanol solvent percentage and Si-Au volume ratio. As an example, even relatively small 40-nm core-satellites (34 nm Si core + 4 nm Au shell) provided a much red shifted peak centered around 610 nm and having a large tail over 700 nm. The generation of the plasmonic peak is confirmed by modeling of Si@Au core-shells of relevant parameters via Mie theory. Being relatively small and exempt of any toxic impurity due to ultraclean laser synthesis, the Si@Au core-satellites promise a major advancement of imaging and phototherapy modalities based on plasmonic properties of nanomaterials.

Biological Assessment of Laser-Synthesized Silicon Nanoparticles Effect in Two-Photon Photodynamic Therapy on Breast Cancer MCF-7 Cells.

Driven by their distinctive physiological activities, biological properties and unique theranostic modalities, silicon nanoparticles (SiNPs) are one of the promising materials for the development of novel multifunctional nanoplatforms for biomedical applications. In this work, we assessed the possibility to use laser-synthesized Si NPs as photosensitizers in two-photon excited photodynamic therapy (TPE-PDT) modality. Herein, we used an easy strategy to synthesize ultraclean and monodispersed SiNPs using laser ablation and fragmentation sequences of silicon wafer in aqueous solution, which prevent any specific purification step. Structural analysis revealed the spherical shape of the nanoparticles with a narrow size distribution centered at the mean size diameter of 62 nm ± 0.42 nm, while the negative surface charge of -40 ± 0.3 mV ensured a great stability without sedimentation over a long period of time. In vitro studies on human cancer cell lines (breast and liver) and healthy cells revealed their low cytotoxicity without any light stimulus and their therapeutic potential under TPE-PDT mode at 900 nm with a promising cell death of 45% in case of MCF-7 breast cancer cells, as a consequence of intracellular reactive oxygen species release. Their luminescence emission inside the cells was clearly observed at UV-Vis region. Compared to Si nanoparticles synthesized via chemical routes, which are often linked to additional modules with photochemical and photobiological properties to boost photodynamic effect, laser-synthesized SiNPs exhibit promising intrinsic therapeutic and imaging properties to develop advanced strategy in nanomedicine field.

In vivo evaluation of safety, biodistribution and pharmacokinetics of laser-synthesized gold nanoparticles.

Capable of generating plasmonic and other effects, gold nanostructures can offer a variety of diagnostic and therapy functionalities for biomedical applications, but conventional chemically-synthesized Au nanomaterials cannot always match stringent requirements for toxicity levels and surface conditioning. Laser-synthesized Au nanoparticles (AuNP) present a viable alternative to chemical counterparts and can offer exceptional purity (no trace of contaminants) and unusual surface chemistry making possible direct conjugation with biocompatible polymers (dextran, polyethylene glycol). This work presents the first pharmacokinetics, biodistribution and safety study of laser-ablated dextran-coated AuNP (AuNPd) under intravenous administration in small animal model. Our data show that AuNPd are rapidly eliminated from the blood circulation and accumulated preferentially in liver and spleen, without inducing liver or kidney toxicity, as confirmed by the plasmatic ALAT and ASAT activities, and creatininemia values. Despite certain residual accumulation in tissues, we did not detect any sign of histological damage or inflammation in tissues, while IL-6 level confirmed the absence of any chronic inflammation. The safety of AuNPd was confirmed by healthy behavior of animals and the absence of acute and chronic toxicities in liver, spleen and kidneys. Our results demonstrate that laser-synthesized AuNP are safe for biological systems, which promises their successful biomedical applications.

Fabrication of Stable Nanofiber Matrices for Tissue Engineering via Electrospinning of Bare Laser-Synthesized Au Nanoparticles in Solutions of High Molecular Weight Chitosan.

We report a methodology for the fabrication of neutralized chitosan-based nanofiber matrices decorated with bare Au nanoparticles, which demonstrate stable characteristics even after prolonged contact with a biological environment. The methodology consists of electrospinning of a mixture of bare (ligand-free) laser-synthesized Au nanoparticles (AuNPs) and solutions of chitosan/polyethylene oxide (ratio 1/3) containing chitosan of a relatively high molecular weight (200 kDa) and concentration of 3% (w/v). Our studies reveal a continuous morphology of hybrid nanofibers with the mean fiber diameter of 189 nm ± 86 nm, which demonstrate a high thermal stability. Finally, we describe a protocol for the neutralization of nanofibers, which enabled us to achieve their structural stability in phosphate-buffered saline (PBS) for more than six months, as confirmed by microscopy and FTIR measurements. The formed hybrid nanofibers exhibit unique physicochemical properties essential for the development of future tissue engineering platforms.

Laser- synthesized TiN nanoparticles as promising plasmonic alternative for biomedical applications.

Exhibiting a red-shifted absorption/scattering feature compared to conventional plasmonic metals, titanium nitride nanoparticles (TiN NPs) look as very promising candidates for biomedical applications, but these applications are still underexplored despite the presence of extensive data for conventional plasmonic counterparts. Here, we report the fabrication of ultrapure, size-tunable TiN NPs by methods of femtosecond laser ablation in liquids and their biological testing. We show that TiN NPs demonstrate strong and broad plasmonic peak around 640-700 nm with a significant tail over 800 nm even for small NPs sizes (<7 nm). In vitro tests of laser-synthesized TiN NPs on cellular models evidence their low cytotoxicity and excellent cell uptake. We finally demonstrate a strong photothermal therapy effect on U87-MG cancer cell cultures using TiN NPs as sensitizers of local hyperthermia under near-infrared laser excitation. Based on absorption band in the region of relative tissue transparency and acceptable biocompatibility, laser-synthesized TiN NPs promise the advancement of biomedical modalities employing plasmonic effects, including absorption/scattering contrast imaging, photothermal therapy, photoacoustic imaging and SERS.

Recent Advances in Laser-Ablative Synthesis of Bare Au and Si Nanoparticles and Assessment of Their Prospects for Tissue Engineering Applications.

Driven by surface cleanness and unique physical, optical and chemical properties, bare (ligand-free) laser-synthesized nanoparticles (NPs) are now in the focus of interest as promising materials for the development of advanced biomedical platforms related to biosensing, bioimaging and therapeutic drug delivery. We recently achieved significant progress in the synthesis of bare gold (Au) and silicon (Si) NPs and their testing in biomedical tasks, including cancer imaging and therapy, biofuel cells, etc. We also showed that these nanomaterials can be excellent candidates for tissue engineering applications. This review is aimed at the description of our recent progress in laser synthesis of bare Si and Au NPs and their testing as functional modules (additives) in innovative scaffold platforms intended for tissue engineering tasks.

Bare laser-synthesized Au-based nanoparticles as nondisturbing surface-enhanced Raman scattering probes for bacteria identification.

The ability of noble metal-based nanoparticles (NPs) (Au, Ag) to drastically enhance Raman scattering from molecules placed near metal surface, termed as surface-enhanced Raman scattering (SERS), is widely used for identification of trace amounts of biological materials in biomedical, food safety and security applications. However, conventional NPs synthesized by colloidal chemistry are typically contaminated by nonbiocompatible by-products (surfactants, anions), which can have negative impacts on many live objects under examination (cells, bacteria) and thus decrease the precision of bioidentification. In this article, we explore novel ultrapure laser-synthesized Au-based nanomaterials, including Au NPs and AuSi hybrid nanostructures, as mobile SERS probes in tasks of bacteria detection. We show that these Au-based nanomaterials can efficiently enhance Raman signals from model R6G molecules, while the enhancement factor depends on the content of Au in NP composition. Profiting from the observed enhancement and purity of laser-synthesized nanomaterials, we demonstrate successful identification of 2 types of bacteria (Listeria innocua and Escherichia coli). The obtained results promise less disturbing studies of biological systems based on good biocompatibility of contamination-free laser-synthesized nanomaterials.

Cavitation-Free Continuous-Wave Laser Ablation from a Solid Target to Synthesize Low-Size-Dispersed Gold Nanoparticles.

Continuous wave (CW) radiation from a Yb-fiber laser (central wavelength 1064 nm, power 1-200 W) was used to initiate ablation of a gold target in deionized water and to synthesize bare (unprotected) gold nanoparticles. We show that the formed nanoparticles present a single low-size-dispersed population with a mean size of the order of 10 nm, which contrasts with previously reported data on dual populations of nanoparticles formed during pulsed laser ablation in liquids. The lack of a second population of nanoparticles is explained by the absence of cavitation-related mechanism of material ablation, which typically takes place under pulsed laser action on a solid target in liquid ambience, and this supposition is confirmed by plume visualization tests. We also observe a gradual growth of mean nanoparticle size from 8-10 nm to 20-25 nm under the increase of laser power for 532 nm pumping wavelength, whereas for 1064 nm pumping wavelength the mean size 8-10 nm is independent of radiation power. The growth of the nanoparticles observed for 532 nm wavelength is attributed to the enhanced target melting and splashing followed by additional heating due to an efficient excitation of plasmons over gold nanoparticles. Bare, low-size-dispersed gold nanoparticles are of importance for a variety of applications, including biomedicine, catalysis, and photovoltaics. The use of CW radiation for nanomaterial production promises to improve the cost efficiency of this technology.

Ultrapure laser-synthesized Si-based nanomaterials for biomedical applications: in vivo assessment of safety and biodistribution.

Si/SiOx nanoparticles (NPs) produced by laser ablation in deionized water or aqueous biocompatible solutions present a novel extremely promising object for biomedical applications, but the interaction of these NPs with biological systems has not yet been systematically examined. Here, we present the first comprehensive study of biodistribution, biodegradability and toxicity of laser-synthesized Si-SiOx nanoparticles using a small animal model. Despite a relatively high dose of Si-NPs (20 mg/kg) administered intravenously in mice, all controlled parameters (serum, enzymatic, histological etc.) were found to be within safe limits 3 h, 24 h, 48 h and 7 days after the administration. We also determined that the nanoparticles are rapidly sequestered by the liver and spleen, then further biodegraded and directly eliminated in urine without any toxicity effects. Finally, we found that intracellular accumulation of Si-NPs does not induce any oxidative stress damage. Our results evidence a huge potential in using these safe and biodegradable NPs in biomedical applications, in particular as vectors, contrast agents and sensitizers in cancer therapy and diagnostics (theranostics).

Ultrapure laser-synthesized Si nanoparticles with variable oxidation states for biomedical applications.

We employ a method of femtosecond laser fragmentation of preliminarily prepared water-dispersed microcolloids to fabricate aqueous solutions of ultrapure bare Si-based nanoparticles (Si-NPs) and assess their potential for biomedical applications. The nanoparticles appear spherical in shape, with low size dispersion and a controllable mean size, from a few nm to several tens of nm, while a negative surface charge (-35 mV ± 0.10 according to z-potential data) provides good electrostatic stabilization of colloidal Si-NP solutions. Structural analysis shows that the Si-NPs are composed of Si nanocrystals with inclusions of silicon oxide species, covered by a SiOx (1 < x < 2) shell, while the total oxide content depends on whether the fragmentation is performed in normal oxygen-saturated water (oxygen-rich conditions) or in water deoxygenated by pumping with noble gases (Ag or He) before and during the experiment (oxygen-free conditions). Our dissolution tests show the excellent water-solubility of all the NPs, while more oxidized NPs demonstrate much faster dissolution kinetics, which is explained by oxidation-induced defects in the core of the Si-NPs. Finally, by examining the interaction of the NPs with human cells after 72 h of incubation at different concentrations, we report the absence of any adverse effects of the NPs up to high concentrations (50 µg mL-1) and a good internalization of NPs via a classical endocytosis mechanism. Possessing far superior purity compared to their chemically synthesized counterparts and enabling a variety of imaging and therapeutic functionalities, the laser-synthesized Si-NPs are promising for safe and efficient applications in nanomedicine.

Characterization of materials released into water from paint containing nano-SiO2.

In order to assess the possible risks of applications containing engineered nanomaterials, it is essential to generate more data about their release and exposure, so far largely overlooked areas of research. The aim of this work was to study the characterization of the materials released from paint containing nano-SiO2 during weathering and exposure to water. Panels coated with nano-SiO2 containing paint and a nano-free reference paint were exposed to accelerated weathering cycles in a climate chamber. The total release of 89 six-hour cycles of UV-illumination and precipitation was 2.3% of the total SiO2 contained in the paint. Additional tests with powdered and aged paint showed that the majority of the released Si was present in dissolved form and that only a small percentage was present in particulate and nano-particulate form. TEM imaging of the leachates indicated that the majority of the particulate Si was contained in composites together with Ca, representing the paint matrix, and only few single dispersed SiO2-NPs were detected. The results suggest that toxicological and ecotoxicological studies need to consider that the released particles may have been transformed or are embedded in a matrix.

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications.

Due to excellent biocompatibility, chemical stability, and promising optical properties, gold nanoparticles (Au-NPs) are the focus of research and applications in nanomedicine. Au-NPs prepared by laser ablation in aqueous biocompatible solutions present an essentially novel object that is unique in avoiding any residual toxic contaminant. This paper is conceived as the next step in development of laser-ablated Au-NPs for future in vivo applications. The aim of the study was to assess the safety, uptake, and biological behavior of laser-synthesized Au-NPs prepared in water or polymer solutions in human cell lines. Our results showed that laser ablation allows the obtaining of stable and monodisperse Au-NPs in water, polyethylene glycol, and dextran solutions. The three types of Au-NPs were internalized in human cell lines, as shown by transmission electron microscopy. Biocompatibility and safety of Au-NPs were demonstrated by analyzing cell survival and cell morphology. Furthermore, incubation of the three Au-NPs in serum-containing culture medium modified their physicochemical characteristics, such as the size and the charge. The composition of the protein corona adsorbed on Au-NPs was investigated by mass spectrometry. Regarding composition of complement C3 proteins and apolipoproteins, Au-NPs prepared in dextran solution appeared as a promising drug carrier. Altogether, our results revealed the safety of laser-ablated Au-NPs in human cell lines and support their use for theranostic applications.

Radio frequency radiation-induced hyperthermia using Si nanoparticle-based sensitizers for mild cancer therapy.

Offering mild, non-invasive and deep cancer therapy modality, radio frequency (RF) radiation-induced hyperthermia lacks for efficient biodegradable RF sensitizers to selectively target cancer cells and thus avoid side effects. Here, we assess crystalline silicon (Si) based nanomaterials as sensitizers for the RF-induced therapy. Using nanoparticles produced by mechanical grinding of porous silicon and ultraclean laser-ablative synthesis, we report efficient RF-induced heating of aqueous suspensions of the nanoparticles to temperatures above 45-50 °C under relatively low nanoparticle concentrations (<1 mg/mL) and RF radiation intensities (1-5 W/cm(2)). For both types of nanoparticles the heating rate was linearly dependent on nanoparticle concentration, while laser-ablated nanoparticles demonstrated a remarkably higher heating rate than porous silicon-based ones for the whole range of the used concentrations from 0.01 to 0.4 mg/mL. The observed effect is explained by the Joule heating due to the generation of electrical currents at the nanoparticle/water interface. Profiting from the nanoparticle-based hyperthermia, we demonstrate an efficient treatment of Lewis lung carcinoma in vivo. Combined with the possibility of involvement of parallel imaging and treatment channels based on unique optical properties of Si-based nanomaterials, the proposed method promises a new landmark in the development of new modalities for mild cancer therapy.

Behavior of TiO(2) released from Nano-TiO(2)-containing paint and comparison to pristine Nano-TiO(2).

In the assessment of the fate and effects of engineered nanomaterials (ENM), the current focus is on studying the pristine, unaltered materials. However, ENM are incorporated into products and are released over the whole product life cycle, though mainly during the use and disposal phases. So far, released ENMs have only been characterized to a limited extent and almost nothing is known about the behavior of these materials under natural conditions. In this work we obtained material that was released from aged paint containing nano-TiO2, characterized the particulate materials, and studied their colloidal stability in media with different pH and ionic composition. A stable suspension was obtained from aged paint powder by gentle shaking in water, producing a dilute suspension of 580 µg/L TiO2 with an average particle size of 200-300 nm. Most particles in this suspension were small pieces of paint matrix that also contained nano-TiO2. Some free nano-TiO2 particles were observed by electron microscopy, but the majority was enclosed by the organic paint binder. The pristine nano-TiO2 showed the expected colloidal behavior with increasing stability with increasing pH and strong agglomeration above the isoelectric point and settling in the presence of Ca. The released TiO2 showed very small variations in particle size, ζ potential, and colloidal stability, even in the presence of 3 mM Ca. The results show that the behavior of released ENM may not necessarily be predicted by studying the pristine materials. Additionally, effect studies need to focus more on the particles that are actually released as we can expect that the toxic effect will also be markedly different between pristine and product released materials.

Novel contributions on luminescent apatite-based colloids intended for medical imaging.

The setup of colloidal hybrid nanosystems based on biomimetic calcium phosphate apatites doped with europium ions has recently raised great interest in the pharmacological community, especially due to their bio-inspired character. This is especially relevant in relation with medical imaging for cancer diagnosis. Questions however remain in relation to a number of applicability aspects, some of which have been examined in this contribution. In a first part of this work, we explored further the luminescence properties of such colloidal nanoparticles. We pointed out, upon excitation of europium, the existence of some non-radiative de-excitation via the vibration of O-H oscillators located at the vicinity of the Eu(3+) luminescent centers. The replacement of Eu(3+) by Tb(3+) ions, less prone to non-radiative de-excitation, was then tested in a preliminary way and can be seen as a promising alternative. In a second part of this work, we inspected the possibility to store these colloids in a dry state while retaining a re-suspension ability preserving the nanometer size of the initial nanoparticles, and we propose a functional protocol involving the addition of glucose prior to freeze-drying. We finally showed for the first time, based on titrations of intracellular Ca(2+) and Eu(3+) ions, that folic acid-functionalized biomimetic apatite nanoparticles were able to target cancer cells that overexpress folate receptors on their membrane, which we point out here in the case of T-47-D breast carcinoma cells, as opposed to ZR-75-1 cells that do not express folate receptors. This contribution thus opens new exciting perspectives in the field of targeted cancer diagnosis, thus confirming the promise of biomimetic apatites-based colloidal formulations.