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Understanding the behavior of multifunctional colloidal nanoparticles capable of biomolecular targeting remains a fascinating challenge in materials science with dramatic implications in view of a possible clinical translation. In several circumstances, assumptions on structure–activity relationships have failed in determining the expected responses of these complex systems in a biological environment. The present Review depicts the most recent advances about colloidal nanoparticles designed for use as tools for cellular nanobiotechnology, in particular, for the preferential transport through different target compartments, including cell membrane, cytoplasm, mitochondria, and nucleus. Besides the conventional entry mechanisms based on crossing the cellular membrane, an insight into modern physical approaches to quantitatively deliver nanomaterials inside cells, such as microinjection and electroporation, is provided. Recent hypotheses on how the nanoparticle structure and functionalization may affect the interactions at the nano–bio interface, which in turn mediate the nanoparticle internalization routes, are highlighted. In addition, some hurdles when this small interface faces the physiological environment and how this phenomenon can turn into different unexpected responses, are discussed. Finally, possible future developments oriented tosynergistically tailor biological and chemical properties of nanoconjugates to improve the control over nanoparticle transport, which could open new scenarios in the fi eld of nanomedicine, are addressed.

Among polymeric nanoparticles designed for cancer therapy, PLGA nanoparticles have become one of the most popular polymeric devices for chemotherapeutic-based nanoformulations against several kinds of malignant diseases. Promising properties, including long-circulation time, enhanced tumor localization, interference with "multidrug" resistance effects, and environmental biodegradability, often result in an improvement of the drug bioavailability and effectiveness. In the present work, we have synthesized 1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (ASC-J9) and developed uniform ASC-J9-loaded PLGA nanoparticles of about 120 nm, which have been prepared by a single-emulsion process. Structural and morphological features of the nanoformulation were analyzed, followed by an accurate evaluation of the in vitro drug release kinetics, which exhibited Fickian law diffusion over 10 days. The intracellular degradation of ASC-J9-bearing nanoparticles within estrogen-dependent MCF-7 breast cancer cells was correlated to a time- and dose-dependent activity of the released drug. A cellular growth inhibition associated with a specific cell cycle G2/M blocking effect caused by ASC-J9 release inside the cytosol allowed us to put forward a hypothesis on the action mechanism of this nanosystem, which led to the final cell apoptosis. Our study was accomplished using Annexin V-based cell death analysis, MTT assessment of proliferation, radical scavenging activity, and intracellular ROS evaluation. Moreover, the intracellular localization of nanoformulated ASC-J9 was confirmed by a Raman optical imaging experiment designed ad hoc. PLGA nanoparticles and ASC-J9 proved also to be safe for a healthy embryo fibroblast cell line (3T3-L1), suggesting a possible clinical translation of this potential nanochemotherapeutic to expand the inherently poor bioavailability of hydrophobic ASC-J9 that could be proposed for the treatment of malignant breast cancer. © 2014 American Chemi

We present a facile, one-pot procedure for the organic-to-water phase transfer and biofunctionalization of semiconductor nanocrystals (quantum dots, or QDs) which employs a synthetic functional copolymer, namely poly(DMA-NAS-MAPS), consisting of three components: a surface interacting monomer, N,N-dimethylacrylamide (DMA), a chemically reactive monomer, N-acryloyloxysuccinimide (NAS), and a silane monomer, [3-(methacryloyloxy)-propyl]-trimethoxysilane (MAPS). The nanocrystals were transferred to water by exploiting the amphiphilic character of the copolymer backbone. Hydrolyzed MAPS units contributed to improve the solubility of QDs in water, whereas NAS exhibited reactivity toward biomolecules. A solution of streptavidin in phosphate buffer exhibited good dispersion ability leading to a clear and transparent colloidal suspension, indicative of good QD dispersion during phase transfer and purification. Unlike most of the published methods, the proposed functionalization approach does not require coupling agents and multistep reactions

Nanomedicine has emerged in the past decade as a promising tool for several therapeutic and diagnostic applications. The development of nanoconjugates containing bioactive ligands specific for targeting cancer cell receptors has become a primary objective of modern nanotechnology. The design of ideal nanoconjugates requires optimization of fundamental parameters including size, shape, ligand shell composition, and reduction in nonspecific protein adsorption. Of great importance is the choice of bioconjugation approach, given that it affects the orientation, accessibility, and bioactivity of the targeting molecule. We provide an overview of recent advances in the immobilization of targeting proteins, focusing on methods to control ligand orientation and density, and highlight criteria for nanoparticle design and development required to achieve enhanced receptor-targeting efficiency. © 2013 Elsevier Ltd.

In this paper we present a new protocol for the synthesis of Star-Like Gold Nanoparticles (SGNs) by a simple one-step, room temperature procedure not involving the use of seeds or surfactants, that can be performed in seconds in any laboratory without the need of special technologies. These particles exhibited excellent properties for Surface Enhanced Raman Spectroscopy (SERS) and, when compared with spherical nanoparticles with similar size and concentration, showed enhancing factors from 10 to 50 times higher depending on the dye and on the wavelength employed. SGNs could be used directly in suspension as single, non-aggregating particles and were shown to be active in a remarkably broad range of the light spectrum from green to near infrared. Moreover, SGNs were adsorbed on the surface of a silicon slide to prepare SERS active solid substrate. Despite the fact that the surface of the solid substrate was not perfectly homogeneous, the signals recorded from different positions acquired through DuoScan averaging mode show excellent reproducibility, demonstrating how this simple and cheap protocol can be applied in order to generate reliable and homogeneous SERS substrates

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the biologically mediated site-specific covalent conjugation of targeting proteins to nanoparticles. ScFv800E6, an scFv mutant selective for HER2 antigen overexpressed in breast cancer cells was chosen as targeting ligand. The fusion protein SNAP-scFv was irreversibly immobilized on magnetofluorescent nanoparticles through the recognition between SNAP module and pegylated O 6-alkylguanine derivative. The targeting efficiency of the resulting nanoparticle against HER2-positive breast cancer cells was assessed by flow cytometry and immunofluorescence. © 2013 Copyright SPIE.

Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests. © 2013 Copyright SPIE.

T A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their in vivo localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct.

Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, lowcost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

The surface activation of multifunctional nanoparticles (MNPs) with peptide ligands directing their targeting to cancer cells is an emerging research land in nanobiotechnology. In this paper, water-soluble MNPs have been synthesized and functionalized with an scFv antibody variant specific toward HER2 receptor overexpressed in several breast cancer cell lines. The scFv was genetically engineered to introduce a cysteine residue inside the loop sequence bridging the VH and VL lobes of the molecule and a histidine tag at the C-terminus in the VL fragment. The Cys and 6×His functionalities were exploited as orthogonal reactive groups driving the scFv conjugation to MNPs. In this way, scFv positioning on MNP surface was forced in two different orientations depending on the molecular binding site used for conjugation. The resulting scFv-functionalized MNP1 and MNP2, respectively, were assessed as to their labeling efficiency and selectivity against HER2-positive MCF7 cells. We demonstrate that, while both MNP1 and MNP2 were selective for HER2, there is a remarkable preference for scFv presentation with VH and VL lobes concurrently available for receptor recognition (MNP1) in terms of cell binding efficiency suggesting that ligand orientation may strongly affect cell-binding efficiency from MNPs

Paper-based SERS active substrates were prepared adsorbing spherical and star-shaped gold nanoparticles on a standard filter paper support. Besides the deposition conditions, morphological parameters of the particles were found to strongly affect the enhancer properties of the substrates. The developed substrate was tested regarding surface homogeneity as well as in the quantitative analysis of malachite green, - a well documented Raman reporter dye - and proved to be capable also to detect the oxidation products of apomorphine, a well-known drug molecule used in Parkinson's disease. This material is simple to prepare, easy to handle and dispose and as such it could be a perfect target for further development of a new family of mass-produced, cheap solid SERS substrates. © 2013 Elsevier B.V.

In control: The ligand positioning and orientation on multifunctional nanoparticles (MNP) are controlled by using a versatile bimodular approach that exploits an 11 amino acid (U11) homing peptide genetically fused with a haloalkane dehalogenase (HALO) enzyme. The resultant nanoparticles have good targeting efficiency and selectivity toward broadly occurring urokinase plasminogen activator receptor positive human cancer cells. Copyright 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PLGA nanoparticles are among the most studied polymer nanoformulations for several drugs against different kinds of malignant diseases, thanks to their in vivo stability and tumor localization exploiting the well-documented "enhanced permeation and retention" (EPR) effect. In this paper, we have developed uniform curcumin-bearing PLGA nanoparticles by a single-emulsion process, which exhibited a curcumin release following a Fickian-law diffusion over 10 days in vitro. PLGA nanoparticles were about 120 nm in size, as determined by dynamic light scattering, with a surface negative charge of -30 mV. The loading ratio of encapsulated drug in our PLGA nanoformulation was 8 wt%. PLGA encapsulation provided efficient protection of curcumin from environment, as determined by fluorescence emission experiments. Next, we have investigated the possibility to study the intracellular degradation of nanoparticles associated with a specific G2/M blocking effect on MCF7 breast cancer cells caused by curcumin release in the cytoplasm, which provided direct evidence on the mechanism of action of our nanocomplex. This study was carried out using Annexin V-based cell death analysis, MTT assessment of proliferation, flow cytometry, and confocal laser scanning microscopy. PLGA nanoparticles proved to be completely safe, suggesting a potential utilization of this nanocomplex to improve the intrinsically poor bioavailability of curcumin for the treatment of severe malignant breast cancer.

The early identification of the insurgence of a malignant cancer and the selective targeting of the tumor with specific drugs are still an open frontier for cancer diagnosis and treatment. The ultimate goal is to improve the therapy efficiency and to reduce the side effects usually encountered with conventional chemotherapy. Worldwide, mammary carcinoma represents the second most recurrent type of malignant tumor in adult women and the fifth cause of death among cancer types. In the context of this thesis, I have designed and developed multifunctional hybrid nanoparticles consisting of an inorganic iron oxide core, useful as source of signal for magnetic resonance imaging (MRI), and an organic shell, including bioactive ligands for the pharmacological effect combined with specific cell targeting, and a molecular dye as fluorescence signal emitter. The nanoparticle characteristics were optimized in terms of size, morphology, surface charge, stability, fluorescence emission and capability to enhance the MRI contrast. In addition, specific biomolecular ligands based on anti-HER-2 monoclonal antibody have been developed and novel strategies for their conjugation to nanoparticles were explored. The resulting hybrid nanocomplexes were tested both in vitro and in vivo to evaluate their toxicity, endocytosis, degradation pathways, and the efficient recognition of cell-surface biomarkers. Next, these nanoparticles proved to be highly effective in selectively targeting breast cancer cells in transplanted mice bearing HER-2-positive tumors. A multifaceted bioanalytical approach, combining fluorescence, magnetic relaxivity, transmission electron microscopy, and histological experiments in vivo and ex vivo, has demonstrated that these nanoprobes prevalently accumulated at the tumor by an active targeting route. The nanoparticles were endocytosed by the tumor cells following a lysosomal pathway of degradation, while did not result in permanent damage of healthy tissues. The princ

A basic overview of essential aspects of the very rich chemistry of boron is discussed to elucidate the structural behavior and chemical properties of boron-containing molecules. Some introductory elements are provided concerning the peculiar chemical properties of boron, which derive from its electronic configuration, bonding orbitals, and electronegativity, and are fundamental to understanding boron's behavior. The structural complexity deriving from such properties, which results in a variety of structures of elemental boron and boron derivatives, will be subsequently introduced, followed by an outline on the main classes of boron-containing compounds. This overview will begin with inorganic boron derivatives, followed by carbon-containing and, finally, organic boron compounds

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the site-specific covalent conjugation of targeting peptides to nanoparticles. An scFv mutant selective for HER2 tumor antigen is chosen as the targeting ligand. SNAP-scFv is immobilized on magnetofluorescent nanoparticles and its targeting efficiency against HER2-positive cells is assessed by flow cytometry and immunofluorescence. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

All bound up: A small variant of protein A (red; see picture) was used as a biotemplate for the synthesis and biofunctionalization of gold nanoparticles (AuNPs). These functionalized AuNPs were able to bind to antibodies (green blocks, trastuzumab; TZ) with a defined orientation, thus showing promise as bio-nanoparticle systems suitable for selective cell labeling by membrane-receptor-specific recognition.

In this review an overview about biological applications of magnetic colloidal nanoparticles will be given, which comprises their synthesis, characterization, and in vitro and in vivo applications. The potential future role of magnetic nanoparticles compared to other functional nanoparticles will be discussed by highlighting the possibility of integration with other nanostructures and with existing biotechnology as well as by pointing out the specific properties of magnetic colloids. Current limitations in the fabrication process and issues related with the outcome of the particles in the body will be also pointed out in order to address the remaining challenges for an extended application of magnetic nanoparticles in medicine.

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.

The exciting aptitude of sugars to form high-affinity interactions with various biological entities exploiting multivalency has led to the assessment of the potential of conjugating mono- and oligosaccharides to nanopartieles to investigate the biorecognition events and interfere with biological processes involving carbohydrates. Since a critical step in these studies is the fine control on the presentation of the saccharide molecules, reliable methods to check their presence and, possibly, their arrangement on the surface of nanopartieles are urgently required. In this chapter, we provide an overview of the most common analytical techniques useful to characterize the saccharidic portion of glycoconjugate nanomaterials. Among them, HRMAS NMR looks particularly promising, as it can resolve the structures of the immobilized ligands and may provide information on their orientation on the nanoparticle surface

In this paper, a straightforward method based on elastic light scattering is shown to provide a sensitive and reliable tool for the quantitative determination of receptor-ligand interactions that occur at the surface of suitably designed core-shell nanoparticles. The assay makes use of monodisperse nanocolloids that have minimal optical contrast with the aqueous environment. By properly coating the particles with an appropriate receptor and unreactive amphiphiles, we developed a hybrid system that combines the availability of standard ligands with the necessary bioinvisibility towards the accidental adsorption of nonspecific macromolecules. This system has been used for the study of the interactions between small molecules (Vancomycin-Lipid 2), and between proteins (Avidin-biotinnylated IgG. © 2011 IEEE.

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface. © 2011 American Chemical Society.

Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.

We present the synthesis of trastuzumab-functionalized pegylated iron oxide nanoparticles and provide an FTIR-based approach to gain a direct evidence of the actual conservation of the native structure of conjugated antibody. Their target-selectivity to specific cancer cell receptors has been also assessed

Amphiphilic bile acids linked through an oligoethylene glycol to a biotin moiety were synthesized and shown to create micellar structures in aqueous environment, interact with avidin and be efficiently incorporated into hepatocyte cells, suggesting their potential as a drug delivery system against liver diseases. © 2011 The Royal Society of Chemistry.

Taking its toll: LPS-coated magnetic nanoparticles (LMNPs) are synthesized by noncovalently linking LPS to oleylamine-coated iron oxide nanoparticles. LMNPs are stable, water-soluble, have homogeneous size and shape, and load an average of 130 LPS molecules. LMNPs induce mild CD14-dependent Toll-like receptor 4 (TLR4) activation in innate immune cells (see picture). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH(2) initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADE!, for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AAMB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two-electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enha

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH(2) initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADE!, for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AAMB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two-electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enha

Silica-encapsulated iron oxide composite nanoparticles were synthesized and characterized in terms of morphological and physico-chemical properties. These nanoparticles exhibited both fluorescent and magnetic properties useful for labeling of breast cancer cells. The mechanism of uptake by tumor cells, the pathway of degradation and the potential toxicity of these magnetofluorescent nanoparticles were investigated, suggesting that they could be developed as an efficient and safe bimodal contrast agent for detection of breast cancer cells. © 2010 American Institute of Physics.

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin-dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH2 initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH2 for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N-benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two-electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA-MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of twoelectron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhance

Highly fluorinated materials are being widely investigated due to a number of peculiar properties, which are potentially useful for various applications, including use as lubricants, anti-adhesive films, and substitutes for biological fluids for biomedical utilization. However, at present such potential is still poorly exploited. One of the major drawbacks that hampers the rapid development of nanoscale fluoro-hybrid devices is the remarkable inertness of perfluor-polymeric materials that lack reactive functionalities, as they do not offer any functional groups that can be employed to covalently anchor organic molecules on their surface. In this paper, a convenient method for the stable biofunctionalization of strongly unreactive perfluoropolymer nanoparticles (PnPs) is reported. PnPs are easily coated with newly synthesized asymmetric diacetylenic monomer compounds (ADMs), thanks to PnP's high propensity to interact with hydrophobic moieties. Once monomerically adsorbed onto PnPs, such suitably designed ADMs enable the formation of a robust polymeric shell around the perfluoroelastomer core via a clean UV-promoted localized photopolymerization. Given the peculiar optical characteristics of PnPs, the coating of the particles can be monitored step by step using light scattering, which also allows estimation of the fraction of reacted monomers by competitive adsorption with smaller particles. The potential of this method for the biofunctionalization of PnPs is demonstrated with representative proteins and carbohydrates. Among them, the extension to avidin biotin technology may broaden the scope and applicability of this strategy to potentially a large number of molecules of biomedical interest.

Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T2 contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor. © 2010 American Chemical Society.

At present, mammary carcinoma is the second most common type of malignant tumor in adult women after lung cancer, as more than one million women are diagnosed with breast cancer every year. Despite advances in diagnosis and treatment, which have resulted in a decrease in mortality in recent decades, breast cancer remains a major public health problem. One of the most significant unresolved clinical and scientific problems is the occurrence of resistance to clinical treatments and their toxicity (and how to predict, prevent and overcome them). However, the heterogeneity of human breast cancer in terms of genetic features, molecular profiles and clinical behavior represents a constraint obstructing the discovery of a solution to the disease. It is currently considered that the chances of success of therapy may increase if the tumor cells are selectively removed before they can evolve to their mature stages up to metastases production. Therefore, novel and more sensitive diagnostic tools are being developed, with the aim of improving the early and noninvasive detection of rising malignancies and the accuracy of tumor tissue localization. Meanwhile, there is an emerging use of targeted therapies in oncology, depending on the expression of specific proteins or genes present in tumor cells. Among the molecular targets considered for the treatment of breast cancer cells so far, we chose to focus on examples involving overexpression and/or gene amplification of "Human Epidermal growth factor Receptor 2" (HER2) protein. In current studies, various types of nanoparticles conjugated with the anti-HER2 monoclonal antibody, the so-called "trastuzumab", are investigated extensively due to promising results in biological and preclinical applications aimed at improving the treatment of breast cancer. In this paper, we present a critical review of the preparation and use of different kinds of trastuzumab-functionalized nanoparticles, with an emphasis on the therapeutic and diagnosti

An elastic light scattering based method, making use of phantom nanoparticles as substrate organizer, allowed the quantitative evaluation of the molecular recognition events between a series of inactivated mutants of phospholipase D and lysophosphatidylcholine, highlighting remarkable binding capability modifications by single amino acid substitutions.

The development of nanosystems applied to rapid and sensitive measurement of biomarkers in fluid samples is a current major goal in diagnostic biomedicine. In this article, we report the accurate and reliable detection of anti-HSA (human serum albumin) antibodies by protein-functionalized magnetic nanospherical probes due to the reversible alteration of their microaggregation state induced by protein antibody-specific interaction, sensed as changes in the T2 relaxation time of surrounding water molecules. Once the optimal parameters were adjusted, the method proved to be very sensitive, providing concentration- and time-dependent responses. Furthermore, we demonstrate that the developed immunoassay is able to quantitatively determine the biomarker concentration from T2 linear correlation, thereby supplying a rapid, yet accurate, assay with sensitivity in the femtomolar range. The high susceptibility and stability of these magnetic nanoparticles, as well as their accessible synthetic preparation, make these nanosensors a promising new tool for versatile and effective medical diagnostics.

A versatile synthetic platform for the efficient immobilization of PNAs on magnetic iron oxides, providing magnetic nanosensors for selective DNA recognition, is presented.

Managing Malaria. Malaria is responsible for two million deaths per year particularly in developing countries, therefore there is great need for the development of cost effective treatment. The synthesis of a library of trisubstituted triazines with potent antimalarial in vitro activity is reported. Among them, five products may be developed as potential leads in the search for new drugs against plasmodial chloroquine-resistant strains.

We have developed an efficient conversion of amino iron oxides to carbohydrate and protein derived nanoparticles with highly conserved bioactivity through a combination of diazo transfer and azide-alkyne click technology.