This PDF file contains the front matter associated with SPIE Proceedings Volume 8232, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

In this paper we present an overview of our recent studies regarding the interactions of functional nanoparticles with the human umbilical endothelial cells (HUVECs). Cellular uptake, cytotoxicity and laser hyperthermia of cells loaded with gold nanoparticles are discussed. Particles with different shape, size and charge are compared and evaluated to conclude at the most appropriate types for specific biomedical applications (i.e. drug delivery, laser hyperthermia).

Neurons display dendritic spines plasticity and morphology anomalies in numerous psychiatric and neurodegenerative diseases. These changes are associated to abnormal dendritic traffic that can be evidenced by fluorescence microscopy. As a fluorescent probe we propose to use fluorescent diamond nanoparticles with size of < 50 nm. Color centers embedded inside the diamond nanoparticles are perfectly photostable emitters allowing for long-term tracking. Nanodiamond carbon surface is also well suited for biomolecule functionalization to target specific cellular compartments. We show that fluorescent nanodiamonds can be spontaneously internalized in neurons in culture and imaged by confocal and Total Internal Reflection (TIRF) microscopy with a high signal over background ratio.

Cells maintain their homeostatic functions by dynamically regulating the concentrations of intracellular ions via ionic channels. Using two recently developed QD-based nanobiosensors for Cl^- and Na⁺ that emit separate wavelengths, herein we report the fluorescence microscopy measurements of the concentrations of intracellular chloride ([Cl^-]_i) and sodium ([Na+]i) simultaneously in intact cells using a 405 nm excitation wavelength. The Cl-QD₅₂₅ and Na-QD_630nm were constructed by conjugating the chloride ion receptor, MEPTU, and sodium ion receptor, 12-crown-4, within the FRET distances of their respective QD_525nm and QD_630nm. This enables FRET energy transferred from the QD (donor) to the receptor complexes (acceptor). The fluorescence intensities of Cl-QD₅₂₅ and QD_630nm determined by photon counting were inversely proportional to the increased concentrations of the respective Cl^- and Na⁺ according to Stern- Volmer. By co-loading Cl-QD₅₂₅ ^TM and Na-QD₆₃₀ ^TM into HEK-293F or T84 cells, we have determined the dynamic responses of [Cl^-]_i and [Na⁺]_i by pharmacologically manipulating the chloride and sodium channels using their respective agonists and antagonists. The measurements of the dynamics of [Cl^-]_i and [Na⁺]_i have not heretofore been possible without these QD-based nanobiosensors. The predictable physio-pharmacological responses elicited by these agents indicate that the regulatory mechanisms of [Cl^-]_i and [Na⁺]_i are independent but coupled. Investigations into the mechanisms of the [Cl^-]_i and [Na⁺]_i signal transduction pathways and for translational ion channel drug discovery can now be performed using this assay.

MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve therapeutic effect in living animals.

Synaptic vesicles are subcellular organelles that are found in the synaptic bouton and are responsible for the propagation of signals between neurons. Synaptic vesicles undergo endo- and exocytosis with the neuronal membrane to load and release neurotransmitters. Here we discuss how we utilize this property to load nanoparticles as a means of probing the interior of synaptic vesicles. To probe the intravesicular region of synaptic vesicles, we have developed a highly sensitive pH-sensing polymer dot. We feel the robust nature of the pH-sensing polymer dot will provide insight into the dynamics of proton loading into synaptic vesicles.

Currently there is considerable interest in using bioconjugated nanoparticles for in vivo imaging, biosensing and theranostics. Luminescent CdSe/ZnS core shell semiconductor quantum dots (QDs) have unique optical properties and bioconjugation capabilities that make them ideal prototypes for these purposes. We have previously described the metal-affinity association between the imidazole groups of terminal hexahistidine residues of peptides and proteins and the ZnS shell of quantum dots as a useful bioconjugation technique. We have also demonstrated that QDs labeled with an oligohistidine-tagged cell penetrating peptide (CPP) derived from the HIV TAT-protein could undergo specific endocytosis-mediated cellular uptake in both HEK293T/17 and COS-1 cells. However, the QDs were predominantly sequestered in the endosomes. This remains a significant hindrance to future potential cellular imaging applications which require the QDs to access other subcellular organelles. Here we describe the testing of several cytosolic QD delivery modalities including microinjection, the commercial cytosolic delivery agent PULSin, and the cytosolic delivery peptide Palm-1. Palm-1, a palmitylated peptide that is capable of both cellular uptake and rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for cytosolic delivery of QDs. Potential intracellular applications for this peptide are discussed.

Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological conditions.

The interactions between biological systems and nanostructured materials are attracting great interest, due to the possibility to open up novel concepts for the design of smart nano-biomaterials that actively play a functional biological role. On the other hand, the assessment of the potential toxic effects arising from such interactions is gaining increasing attention, and a new field known as nanotoxicology is strongly emerging. In this frame, we investigated the response of human neurons to gold surfaces with different levels of nanoroughness, finding out that neurons are capable to sense and actively respond to these nanotopography features. These nanostructured substrates were also investigated to explore the impact of nanotopography on morphology and genomics of adherent bacteria. A multidisciplinary approach was exploited to characterize bacteria-nanostructured surface interactions, observing that type-1 fimbriae disappear in bacteria grown onto nanorough substrates. We also show how nanoparticles interact with biomolecules in culture media and in vitro and in vivo biological systems, by investigating the toxic effects of a wide range of nanomaterials (AuNPs, QDs, SiO₂ NPs), demonstrating the key role of size, shape, and surface coating.

Using a light-based approach known as photodynamic therapy (PDT), we explore a new method in tackling viral pathogens via the excitation of light-sensitive materials called photosensitizers to produce reactive oxygen species which mediates the inactivation of viruses. Photosensitizers are loaded into mesoporous silica coated upconversion nanoparticles to photodynamically inactivate viruses in suspension.

Cadmium-free quantum dots (QDs), such as those made from InP, show similar optical properties to those containing toxic heavy metals and thus provide a promising alternative for imaging and therapeutics. The band gap of InP is similar to that of CdTe, so photosensitization of InP QDs with porphyrins or other dyes should lead to generation of reactive oxygen species, useful for targeted destruction of malignant cells or pathogenic bacteria. Here we show the results of measurements of singlet oxygen and superoxide generation from InP QDs with single and double ZnS shells compared with CdTe and CdSe/ZnS. Reactive oxygen species are measured using colorimetric or fluorescent reporter assays and spin-trap electron paramagnetic resonance (EPR) spectroscopy. We find that the size of the InP QDs and the thickness of the ZnS shell both strongly influence ROS generation. These results suggest future approaches to the design of therapeutic nanoparticles.

Variety efforts are being made to develop colloidal based drug delivery systems (DDSs), which encapsulate cytotoxic drug in a vehicle and release them in a controlled manner. However, the synthetic carriers developed thus far are hampered by rapidly clearance in the body, for example by phagocytes, possibly due to the non-natural surface characteristics in terms of chemistry, morphology, and mechanics. To circumvent this important challenge, we have exploited living mammalian cells as factories to encapsulate drugs in "natural vesicles". These natural vesicles are termed cell membrane capsules (CMCs), because they maintain the major membrane structure and functions as well as cytosolic proteins of the parental cells. We demonstrate that CMCs act as unique delivery vehicles, in which encapsulated substances can be processed stepwise by cellular enzymes and then be selectively released through protein channels built-in the membrane, in a controlled and sustained manner. The preliminary study investigating the macrophage response to CMCs indicated the potential of CMCs to avoid attack by the immune system.

Magnetic-fluorescent nanoparticles have been emerging as potential bimodal probes in the area of bioimaging. However, near-infrared (NIR) fluorescent dye as a fluorescent material for bimodal probe remains unexplored. The tailor-design of NIR cyanine dye is challenging. Herein, we report the synthesis and characterization of novel functional IR 820 dye. This modified IR 820 has been successfully conjugated with long and short back-bone chain polymers. All these compounds preserve good water solubility and photochemical properties. The magnetic-fluorescent bimodal probe has been demonstrated, wherein the magnetic nanoparticles have been coated with dye-polymer. The cytotoxicity studies on HeLa cells show that MNP@dye-polymer with short back-bone chain has better cell viability.

Iron oxide colloidal nanocrystals (ferrofluids) are investigated for application in the treatment of cystic fibrosis lung infections, the leading cause of mortality in cystic fibrosis patients. We investigate the use of iron oxide nanocrystals to increase the effectiveness of inhalation aerosol antibiotics therapy through two mechanisms: directed particle movement in the presence of a static external magnetic field and magnetic hyperthermia. Magnetic hyperthermia is an effective method for decreasing the viscosity of the mucus and biofilm thereby increasing drug, immune cell, and antibody penetration to the affected area. Iron oxide nanocrystals of various sizes and morphologies were synthesized and tested for specific losses (heating power) using frequencies of 111.1 kHz and 629.2 kHz, and corresponding magnetic field strengths of 9 and 25 mT. Nanocrystals in the superparamagnetic to ferromagnetic size range exhibited excellent heating power. Additionally, iron oxide-zinc selenide core-shell nanoparticles were prepared in parallel in order to allow imaging of the iron oxide nanoparticles.

At the interface with solids, liquid molecules tend to adopt a particular structural arrangement, which depends on the local interplay of forces between the solid and the liquid. As a result, this interfacial liquid does not behave like bulk liquid, often exhibiting higher densification and ordering (solvation structure) that strongly depends on the local properties of the solid surface. Being a link between a solid and its surroundings, the interfacial liquid largely determines how a particular solid will interact with its environment. This is particularly relevant for nanomedicine where synthetic nano-objects are injected in a liquid environment for therapeutic purposes. Experimentally, little is known about the local structure and properties of interfacial liquid due to the lack of techniques offering sufficient resolution over inhomogeneous surfaces. Recently we demonstrated how amplitude-modulation atomic force microscopy (AM-AFM) could be used to probe and quantify solid-liquid interfaces locally and with sub-nanometer resolution. Here we apply this technique to compare the interface formed by selected solids with water, dimethylsulfoxide and 1-hexanol. The results highlight differences in the solvation structure formed by the different liquids over a same solid. Importantly, our results show that AM-AFM images reflect the structure of the interfacial liquid, which is not necessarily following the atomic structure of the interfaced solid in a trivial manner. These findings have important implications for the interpretation of AM-AFM images in liquid.

This paper demonstrates a new method for fabrication of stable gold nanoparticle-poly(ethylene glycol) (PEG) conjugates with a defined number of PEG molecules. The PEG molecules are directly bound to the surface of gold nanoparticles with almost 100% conjugation efficiency and the PEG surface coverage is tunable between values of 0 and 100%. Gold nanoparticles for the nanoparticle PEG conjugates are prepared by femtosecond laser ablation in liquid of a gold bulk target in deionized water. This method for fabrication of nanoparticles creates gold surfaces which are negatively charged and chemically clean. This facilitates uniform and controlled binding of thiolated PEG molecules to the surface of the gold nanoparticles. The method used to bind PEG to gold can be used with other biomolecules to prepare gold nanoparticles with single or mixed monolayers of biologically important molecules.

Lanthanide fluoride colloidal nanocrystals offer a way to improve the diagnosis and treatment of cancer through the enhanced absorption of ionizing radiation, as well as providing visible luminescence. In order to explore this possibility, cytotoxicity assays need to be performed on mammalian cells in vitro, to show minimum levels of biocompatibility for future experiments. 20% lanthanum 60% cerium and 20% europium lanthanide fluoride nanocrystals were capped with polyethylene glycol (PEG) of molecular weight 4000 and suspended in deionized water. These nanocrystals were characterized by transmission electron microscopy, muffle furnace ashing, absorbance spectroscopy, dynamic light scattering, and photoluminescence spectroscopy. Visible light microscopy and trypan blue staining was performed on the cells to assay the cytotoxicity of the nanocrystal on the human astrocytoma line U-87 MG, purchased from ATCC.

Knowledge of the molecular mechanisms underlying the interactions between nanomaterials and living systems is fundamental for providing more effective products for nanomedicine and drug delivery. Controlling the response of cells/bacteria (such as activation of inflammatory processes or apoptosis/necrosis in tumor cells or pathogenic bacteria) by tuning specific properties of the nanomaterials is ultimately the challenging goal. Notably, this may also provide crucial information in the assessment of any toxic risks induced by nanoparticles on humans. However, in studying the nano-biointeractions, it is imperative to take into account the dynamic evolutions of nanoparticles in the biological environments (in terms of protein corona formation, size and charge changes) in synergy with the dynamic events occurring in cells, including signal transduction, metabolic processes, homeostasis and membrane trafficking. In this context, we discuss the impact of analytical technologies, especially in the field of proteomics, which can provide major insights into the processes affecting the NPs surface as well as the cells and bacteria functionalities. In particular, we show that a precise control of the chemical-physical characteristics of the interacting nanoparticles or nanostructures may impact the cells by inducing changes in the proteomic profiles with direct consequences on their viability.

Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

IFN-γ-adsorbed DMSA-coated magnetite nanoparticles can be used as an efficient in vivo drug delivery system for tumor immunotherapy. Magnetic nanoparticles, with adsorbed interferon-γ, were targeted to the tumor site by application of an external magnetic field. A relevant therapeutic dosage of interferon in the tumor was detected and led to a notable reduction in tumor size. In general, only 10% of the total injected nanoparticles after multiple exposures were found in tissues by AC susceptibility measurements of the corresponding resected tissues. Magnetic nanoparticle biodistribution is affected by the application of an external magnetic field.

Magnetic labeling renders cells MRI-detectable which provides attractive solutions for tracking the fate of a transplanted cell population. Understanding the interplay of magnetic nanoparticles and cells is then an important point that should not be neglected. Here we show that in the condition of food starvation, macrophage cells emit vesicles containing nanoparticles. First, we inferred the intracellular iron oxide load from the magnetophoretic velocity of cells at a calibrated magnetic field gradient. After magnetic labeling and culture in stress conditions, the intracellular iron oxide load was once more determined and a detectable difference was observed before and after stress. Moreover, we identified in the stress conditioned medium membrane vesicle structures carrying magnetic particles. Besides pointing out the role of cell-derived vesicles in the sequestration of the intracellular magnetic label, experiments also demonstrated that vesicles were able to chaperone the magnetic cargo into naïve cells.

Magnetite based nanoparticles functionalized with different ligands have been obtained by optimization of two synthetic methods. Gold surrounded Fe₃O₄ nanoparticles capped with oleic acid and oleylamine were achieved by thermal decomposition of metallo-organic precursors. By this way nanoparticles with perfectly defined size within 3.5 nm to 7 nm in diameter and organic content from 16.1% to 40.9 % were obtained. Precipitation of iron(II) chloride in basic solution yield magnetite nanoparticles between 20 and 40 nm with contents of organic ligands of 3 - 12 %. The samples have been characterized by X-ray diffraction, transmission electron microscopy and thermogravimetric measurements. A complete magnetic study has been performed by means of a SQUID magnetometer and electron magnetic resonance (EMR), showing the influence of capping covering on the superparamagnetic behaviour. The citotoxicity and interaction with HeLa cells was evaluated for some of the preparations. Finally, the specific absorption rate (SAR) was calculated to compare the efficiency of heating each sample for the various applied magnetic fields.

Hybrid multifunctional nanoparticles (NPs) are emerging as useful probes for magnetic based targeting, delivery, cell separation, magnetic resonance imaging (MRI), and fluorescence-based bio-labeling applications. Assessing from the literature, the development of multifunctional NPs for multimodality imaging is still in its infancy state. This report focuses on our recent work on quantum dots (QDs), magnetic NPs (MNPs) and bi-functional NPs (composed of either QDs or rare-earth NPs, and magnetic NPs - iron oxide or gadolinium oxide) for multimodality imaging based biomedical applications. The combination of MRI and fluorescence would ally each other in improving the sensitivity and resolution, resulting in improved and early diagnosis of the disease. The challenges in this area are discussed.

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens.

An effective strategy is presented to make spherical Ln³⁺ doped NaYF4 nanoparticles that show upconversion, with the aim of deep-tissue optical imaging. Upconversion is the conversion of two or more low-energy photons into one of higher energy, e.g. 980 nm to 545 and 680 nm and 980 nm to 800 nm. In order to avoid the formation of nanoparticles with an aspect ratio, we developed a strategy in which subsequent shells were grown on spherical seed nanoparticles. The last shell is undoped in order to improve the optical properties. In addition, a simple intercalation strategy involving the oleate ligands on the surface has been developed to make the nanoparticles dispersible in aqueous solutions and physiological buffers. Two-photon upconversion laser scanning microscopy (TPULSM) and two-photon upconversion wide-field microscopy (TPUWFM) have been tested for their suitability in deep-tissue imaging with retention of lateral and depth resolution (also called optical sectioning). TPULSM can be used up to ~ 600 μm deep, but takes inordinately long times to acquire, which is due to the fact that the absorption cross section of Yb³⁺ is low, the quantum yield of the upconversion process are << 1%, and the Ln³⁺ excited states are up to several hundreds of μs. Hence UCNPs in general are not very bright (i.e. large emitted photon flux). The TPUWFM seems more promising because acquisition times are only several minutes, with depth profiling up to 400 μm. We show the first optical sectioning with this technique in the brain of a mouse, through a thin shaved skull.

We present experimental results on the multicolor (blue and green) photoluminescence from glycine-coated silver nanoclusters and small nanoparticles which can be used as novel probes for bio-imaging. Glycine-coated silver nanoclusters and nanoparticles were synthesized using thermal reduction of silver nitrate in a glycine matrix, according to a modified procedure described in literature. The size characterization with mass spectrometry, scanning electron microscopy and dynamic light scattering showed that the diameters of luminescent silver nanoclusters and small nanoparticles vary from 0.5 nm to 17 nm. Extinction spectroscopy revealed that the absorption band of the luminescent nanoclusters and nanoparticles was blue-shifted as compared to the nonluminescent larger silver nanoparticles. This effect indicated the well-known size dependence of the surface plasmon resonance in silver. The most pronounced photoluminescence peak was observed around 410 nm (characteristic SPR wavelength for silver) which strongly suggests the enhancement of the photoluminescence from silver nanoparticles by the SPR. The relative quantum yield of the photoluminescence of silver nanoclusters and nanoparticles was evaluated to be 0.09. In terms of their small size, brightness and photostability, noble metal nanoclusters and nanoparticles hold the most promise as candidates for biological cell imaging, competing with commonly used semiconductor quantum dots, fluorescent proteins and organic dyes. When applied to the problem of intracellular imaging, metal nanoclusters and small nanoparticles offer advantages over their much larger sized semiconductor counterparts in terms of ease of biological delivery. In addition, noble metal nanoparticles and nanoclusters are photostable. The high quantum yield (QY) of the photoluminescence emission signal enables the isolation of their photoluminescence from the cellular autofluorescence in cell imaging, improving the image contrast.

We report design and synthesis of a series of activatable "OFF/ON" CdS:Mn/ZnS quantum dot (Qdot) based sensing probes. The Qdot "OFF" state represent the "quenched state" where the Qdot fluorescence is quenched by ligands attached to Qdot surface. Fluorescence quenching is likely due to ligand assisted electron transfer process. Qdot fluorescence is restored when the electron transfer process is stopped. Using this activatable Qdots, we have successfully demonstrated usefulness of these Qdot probes for reliable detection of toxic cadmium ions in solution, selective detection of glutathione and sensitive detection of intracellular cancer drug release event. In this paper, we will discuss a simple but robust method of making water-soluble CdS:Mn/ZnS Qdots at the room-temperature. Two different water-soluble biomolecules, the N-acetyl cysteine (NAC) and the glutathione (GSH) were used as surface coating ligands. This is a singlestep, one-pot synthesis where the Qdot nanocrystals were grown in the presence of the biomolecules. These Qdots were characterized by fluorescence spectroscopy. Stability of the GSH coated Qdots and the NAC coated Qdots were studied by treating with ethylenediaminetetraacetic acid (EDTA, a strong chelating agent for Zn and Cd ions). Our results show that fluorescence properties of Qdots are affected by the type of surface coated ligands. In comparison to the GSH coated Qdots, the NAC coated Qdots show broad but strong emission towards near infra-red region. When treated with EDTA, fluorescence property of the GSH coated Qdot was affected less than the NAC coated Qdots. This preliminary study shows that NAC coated Qdots could potentially be used to develop activatable ("OFF/ON") probes for potential deep-tissue imaging applications. Similarly, the GSH coated Qdots could be applied for probing desired analytes or for bioimaging purposes in environmentally harsh conditions.

The electronic interaction of semiconductor nanocrystals with adjacent metallic nanostructures is investigated by single nanocrystal fluorescence spectroscopy. We synthesized CdSe multishell semiconductor nanocrystals coated with silica shells and coupled them to self-assembled gold nanoparticle films. The nanocrystals showed an average increase of the on-time photoluminescence intensity by a factor of up to three and a decreased photoluminescence lifetime by about one order of magnitude. In addition, we observed photoluminescence from gray states and a strong blinking suppression with an increase of the on-state fraction from 60% to more than 90%.

Fluorescent metal nanoclusters (NCs) have many attractive features, including ultrasmall size, good biocompatibility and large Stokes shift, thus making them promising alternatives as fluorescent markers for biological applications. We have developed a facile method of synthesizing water-soluble, near-infrared (NIR) luminescent AuNCs. By virtue of the long fluorescence lifetime of these AuNCs, we explored their application in cellular fluorescence lifetime imaging (FLIM). Furthermore, the interaction of AuNCs with biomolecules such as proteins has been investigated. Upon protein association, the fluorescence of AuNCs was found to be enhanced and their luminescence lifetime was prolonged. Our studies underline the importance of studying the behavior of AuNCs within the complex biological environment for their application as novel cellular imaging probes.

Vertically-aligned multi-walled carbon nanotubes (VACNT) is of particular interest in regenerative medicine. Templateinduced hydroxyapatite (HA) has broad prospects in applied fields of bone regenerative medicine. Thus, it becomes very attractive a combination these two excellent materials to bone tissue engineering applications. In this study the HA/VACNT nanocomposites were used as scaffolds to Human osteoblast cells culture. Superhydrophilic VACNT films were obtained by CVD method and funcionalized by oxygen plasma. The fabrication of HA/VACNT nanocomposites was performed with a direct electrodeposition of the thin HA films on the VACNT films. The bioactivity and biomineralization in vitro process of superhydrophilic HA/VACNT nanocomposites were investigated using simulated body fluid (SBF) and optical techniques. The characterization of of HA/VACNT nanocomposites was performed before and after soaking 21 days in SBF and compared to superydrophilic VACNT films. Fourier transform infrared spectroscopy, micro X-ray fluorescence spectrometer by energy-dispersive and X-ray difractogram were employed to investigate the structural and chemical properties. The morphology was investigated by FEG-SEM analysis. After 21 days was identified that others biological apatites were formed only on HA/VACNT nanocomposites. Optical techniques showing a powerful tool to characterizated and investigated the bioactivity in vitro process. These findings were very atractive to application of this new nanocomposite to bone tissue regeneration.

Oleic acid/oleylamine capped Fe_1-xPd_x (x=0.16, 0.20, 0.25) nanoparticles have been synthesized using a diol as reducing agent in order to get nanoparticles of different average sizes and coatings. Composition and morphology were analyzed by infrared spectroscopy (IR), X-Ray Diffraction (RXD), thermogravimetry (TG) and Transmission Electron Microscopy (TEM). Well dispersed nanoparticles with uniform sizes between 3 and 8 nm and average diameters less than 6 nm were obtained. Magnetic properties were investigated by Electron Magnetic Resonance (EMR) and magnetization measurements. Most of the nanoparticles were superparamagnetic at physiological temperature with low magnetic saturation values (near 5 emu.g^-1 alloy/0.4 Nβ per Fe atom). SAR measurements were also performed with the aim of carrying out hyperthermia measurements but the nanoparticles did not work as magnetic heaters.