Synthesis of chemically functionalized superparamagnetic nanoparticles as delivery vectors for chemotherapeutic drugs.

Superparamagnetic iron oxide nanoparticles (SPIONs) are in clinical use for disease detection by MRI. A major advancement would be to link therapeutic drugs to SPIONs in order to achieve targeted drug delivery combined with detection. In the present work, we studied the possibility of developing a versatile synthesis protocol to hierarchically construct drug-functionalized-SPIONs as potential anti-cancer agents. Our model biocompatible SPIONs consisted of an iron oxide core (9-10 nm diameter) coated with polyvinylalcohols (PVA/aminoPVA), which can be internalized by cancer cells, depending on the positive charges at their surface. To develop drug-functionalized-aminoPVA-SPIONs as vectors for drug delivery, we first designed and synthesized bifunctional linkers of varied length and chemical composition to which the anti-cancer drugs 5-fluorouridine or doxorubicin were attached as biologically labile esters or peptides, respectively. These functionalized linkers were in turn coupled to aminoPVA by amide linkages before preparing the drug-functionalized-SPIONs that were characterized and evaluated as anti-cancer agents using human melanoma cells in culture. The 5-fluorouridine-SPIONs with an optimized ester linker were taken up by cells and proved to be efficient anti-tumor agents. While the doxorubicin-SPIONs linked with a Gly-Phe-Leu-Gly tetrapeptide were cleaved by lysosomal enzymes, they exhibited poor uptake by human melanoma cells in culture.

Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells.

Aim: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO-NPs) are under development for imaging and drug delivery; however, their interaction with human blood-brain barrier models is not known. Materials & Methods: The uptake, reactive oxygen species production and transport of USPIO-NPs across human brain-derived endothelial cells as models of the blood-brain tumor barrier were evaluated for either uncoated, oleic acid-coated or polyvinylamine-coated USPIO-NPs. Results: Reactive oxygen species production was observed for oleic acid-coated and polyvinylamine-coated USPIO-NPs. The uptake and intracellular localization of the iron oxide core of the USPIO-NPs was confirmed by transmission electron microscopy. However, while the uptake of these USPIO-NPs by cells was observed, they were neither released by nor transported across these cells even in the presence of an external dynamic magnetic field. Conclusion: USPIO-NP-loaded filopodia were observed to invade the polyester membrane, suggesting that they can be transported by migrating angiogenic brain-derived endothelial cells.

The use of heterogeneous chemistry for the characterization of functional groups at the gas/particle interface of soot and TiO(2) nanoparticles

Six gases (N((CH3)3), NH2OH, CF3COOH, HCl, NO2, O3) were selected to probe the surface of seven combustion aerosol (amorphous carbon, flame soot) and three types of TiO2 nanoparticles using heterogeneous, that is gas-surface reactions. The gas uptake to saturation of the probes was measured under molecular flow conditions in a Knudsen flow reactor and expressed as a density of surface functional groups on a particular aerosol, namely acidic (carboxylic) and basic (conjugated oxides such as pyrones, N-heterocycles) sites, carbonyl (R1-C(O)-R2) and oxidizable (olefinic, -OH) groups. The limit of detection was generally well below 1% of a formal monolayer of adsorbed probe gas. With few exceptions most investigated aerosol samples interacted with all probe gases which points to the coexistence of different functional groups on the same aerosol surface such as acidic and basic groups. Generally, the carbonaceous particles displayed significant differences in surface group density: Printex 60 amorphous carbon had the lowest density of surface functional groups throughout, whereas Diesel soot recovered from a Diesel particulate filter had the largest. The presence of basic oxides on carbonaceous aerosol particles was inferred from the ratio of uptakes of CF3COOH and HCl owing to the larger stability of the acetate compared to the chloride counterion in the resulting pyrylium salt. Both soots generated from a rich and a lean hexane diffusion flame had a large density of oxidizable groups similar to amorphous carbon FS 101. TiO2 15 had the lowest density of functional groups among the three studied TiO2 nanoparticles for all probe gases despite the smallest size of its primary particles. The used technique enabled the measurement of the uptake probability of the probe gases on the various supported aerosol samples. The initial uptake probability, g0, of the probe gas onto the supported nanoparticles differed significantly among the various investigated aerosol samples but was roughly correlated with the density of surface groups, as expected. [Authors]

Differentiated chemical reactivity of nanoparticles toward DTT

RATIONALE: Induction of oxidative stress and impairment of the antioxidant defense are considered important biological responses following nanoparticle (NP) exposure. The acellular in vitro dithiothreitol (DTT) assay is proposed to measure the oxidative potential of NP. In addition, DTT can be considered as a model compound of sulfur containing antioxidants. The objective of this work is to evaluate the surface reactivity in solution of a NP panel toward DTT. METHOD: The NP panel was composed of four carbonaceous particles, six types of metal oxides and silver with primary size ranged from 7 to 300 nm. Suspensions were prepared in surfactant solution with 30 min sonication. DTT was used as reductant to evaluate the oxidative properties of the different NP. The determination of the NP ability to catalyze electron transfer from DTT to oxygen was carried out as described in Sauvain et al., Nanotoxicology, 2008, 2:3, 121−129. RESULTS: All the carbonaceous NP catalyzed the oxidation of DTT by oxygen following the mass based order: carbon black > diesel exhaust particle > nanotubes > fullerene. A contrasting reactivity was observed for the metallic NP. Except for nickel oxide and metallic silver, which reacted similarly to the carbonaceous NP, all other metal oxides hindered the oxidation of DTT by oxygen, with ZnO being the most effective one. CONCLUSIONS : DTT was stabilized against oxidation in the presence of metal oxide NP in the solution. This suggests that different chemical interactions take place compared with carbonaceous NP. To explain these differences, we hypothesize that DTT could form complexes with the metal oxide surface (or dissolved metal ions), rendering it less susceptible to oxidation. By analogy, such a process could be thought to apply in biological systems with sulfur−containing antioxidants, reducing their buffer capacity. Such NP could thus contribute to oxidative stress by an alternative mechanism.

Development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted b diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Benefit of anti-HER2-coated paclitaxel-loaded immuno-nanoparticles in the treatment of disseminated ovarian cancer: Therapeutic efficacy and biodistribution in mice.

The benefit of polymeric immuno-nanoparticles (NPs-Tx-HER), consisting of paclitaxel (Tx)-loaded nanoparticles coated with anti-HER2 monoclonal antibodies (Herceptin, trastuzumab), in cancer treatment was assessed in a disseminated xenograft ovarian cancer model induced by intraperitoneal inoculation of SKOV-3 cells overexpressing HER2 antigens. The study was focused on the evaluation of therapeutic efficacy and biodistribution of NPs-Tx-HER compared to other Tx formulations. The therapeutic efficacy was determined by two methods: bioluminescence imaging and survival rate. The treatment regimen consisted in an initial dose of 20mg/kg Tx administered as 10mg/kg intravenously (IV) and 10mg/kg intraperitonealy (IP), followed by five alternative IP and IV injections of 10mg/kg Tx every 3 days. The bioluminescence study has clearly shown the superior anti-tumor activity of NPs-Tx-HER compared to free Tx. As a confirmation of these results, a significantly longer survival of mice was observed for NPs-Tx-HER treatment compared to free Tx, Tx-loaded nanoparticles coated with an irrelevant mAb (Mabthera, rituximab) or Herceptin alone, indicating the potential of immuno-nanoparticles in cancer treatment. The biodistribution pattern of Tx was assessed on healthy and tumor bearing mice after IV or IP administration. An equivalent biodistribution profile was observed in healthy mice for Tx encapsulated either in uncoated nanoparticles (NPs-Tx) or in NPs-Tx-HER. No significant difference in Tx biodistribution was observed after IV or IP injection, except for a lower accumulation in the lungs when NPs were administered by IP. Encapsulated Tx accumulated in the organs of the reticulo-endothelial system (RES) such as the liver and spleen, whereas free Tx had a non-specific distribution in all tested organs. Compared to free Tx, the single dose injection (IV or IP) of encapsulated Tx in mice bearing tumors induced a higher tumor accumulation. However, no difference in overall tumor accumulation between NPs-Tx-HER and NPs-Tx was observed. In conclusion, the encapsulation of Tx into NPs-Tx-HER immuno-nanoparticles resulted in an improved efficacy of drug in the treatment of disseminated ovarian cancer overexpressing HER2 receptors.

Fate of Hydrophilic Nanoparticles in Biological Environments

The nanoparticles developed are based on chitosan, a biocompatible and biodegradable polysaccharide. The chitosan nanoparticles are formed in an entirely water-based process by electrostatic interactions with other biocompatible molecules. As a prerequisite to understand the fate of such nanoparticles in cells, comprehensive characterization and stability studies serve to identify quantitatively the impact of the raw material characteristics and preparation conditions on the nanoparticle characteristics. Methods included H-1 NMR spectroscopy, dilution viscometry, particle size analysis and electron microscopy. Cytotoxicity and cell uptake experiments on RAW 264.7 murine macrophages and p23 murine endothelial cells were performed to investigate the correlation with nanoparticle characteristics and effect of surface decoration with alginate. Cytotoxicity was assessed by the MTT survival test; cell uptake was monitored by fluorescent microscopy using labeled polymers.

Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.

Nanoparticles for fingermark detection: an insight into the reaction mechanism

This publication presents one of the first uses of silicon oxide nanoparticles to detect fingermarks. The study is not confined to showing successful detection of fingermarks, but is focused on understanding the mechanisms involved in the fingermark detection process. To gain such an understanding, various chemical groups are grafted onto the nanoparticle surface, and parameters such as the pH of the solutions or zeta potential are varied to study their influence on the detection. An electrostatic interaction has been the generally accepted hypothesis of interaction between nanoparticles and fingermarks, but the results of this research challenge that hypothesis, showing that the interaction is chemically driven. Carboxyl groups grafted onto the nanoparticle surfaces react with amine groups of the fingermark secretion. This formation of amide linkage between carboxyl and amine groups has further been favoured by catalyzing the reaction with a compound of diimide type. The research strategy adopted here ought to be applicable to all detection techniques using nanoparticles. For most of them the nature of the interaction remains poorly understood.

Surface-functionalized ultrasmall superparamagnetic nanoparticles as magnetic delivery vectors for camptothecin.

Drug-nanoparticle conjugates: The anticancer drug camptothecin (CPT) was covalently linked at the surface of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) via a linker, allowing drug release by cellular esterases. Nanoparticles were hierarchically built to achieve magnetically-enhanced drug delivery to human cancer cells and antiproliferative activity.The linking of therapeutic drugs to ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) allowing intracellular release of the active drug via cell-specific mechanisms would achieve tumor-selective magnetically-enhanced drug delivery. To validate this concept, we covalently attached the anticancer drug camptothecin (CPT) to biocompatible USPIOs (iron oxide core, 9-10 nm; hydrodynamic diameter, 52 nm) coated with polyvinylalcohol/polyvinylamine (PVA/aminoPVA). A bifunctional, end-differentiated dicarboxylic acid linker allowed the attachment of CPT to the aminoPVA as a biologically labile ester substrate for cellular esterases at one end, and as an amide at the other end. These CPT-USPIO conjugates exhibited antiproliferative activity in vitro against human melanoma cells. The intracellular localization of CPT-USPIOs was confirmed by transmission electron microscopy (iron oxide core), suggesting localization in lipid vesicles, and by fluorescence microscopy (CPT). An external static magnetic field applied during exposure increased melanoma cell uptake of the CPT-USPIOs.

Magmatic fluids in the Breccia-hosted epithermal Au-Ag deposit of Rosia Montana, Romania

The breccia-hosted epithermal Au-Ag deposit of Rosia Montana is located 7 kin northeast of Abrud, in the northern part of the South Apuseni Mountains, Romania. Estimated total reserves of 214.91 million metric toils (Mt) of ore at 1.46 g/t An and 6.9 g/t Ag (10.1 Moz of An and 47.6 Moz of Ag) make Rosia Montana one of the largest gold deposits in Europe. At this location, Miocene calc-alkaline magmatic and hydrothermal activity was associated with local extensional tectonics within a strike-slip regime related to the indentation of the Adriatic microplate into the European plate during the Carpathian orogenesis. The host rocks of the magmatic complex consist of pre-Mesozoic metamorphosed continental crust covered by Cretaceous turbiditic sediment (flysch). Magmatic activity at Rosia Montana and its surroundings occurred in several pulses and lasted about 7 m.y, Rosia Montana is a breccia-hosted epithermal system related to strong phreatomagmatic activity due to the shallow emplacement of the Montana dacite. The Montana dacite intruded Miocene volcaniclastic material (volcaniclastic breccias) and crops out at Cetate and Carnic Hills. Current mining is focused primarily on the Cetate open pit, which was mapped in detail, leading to the recognition of three distinct breccia bodies: the dacite breccia with a dominantly hydrothermal matrix, the gray polymict breccia with a greater proportion of sand-sized matrix support, and the black polymict breccia, which reached to the surface, contains carbonized tree trunks and has a dominantly barren elastic matrix. The hydrothermal alteration is pervasive. Adularia alteration with a phyllic overprint is ubiquitous; silicification and argillic alteration occur locally. Mineralization consists of quartz, adularia, carbonates (commonly Mn-rich), pyrite, Fe-poor sphalerite, galena, chalcopyrite, tetrahedrite, and native gold and occurs as disseminations, as well as in veins and filling vugs within the Montana dacite and the different breccias. The age of mineralization (12.85 +/- 0.07 Ma) was determined by Ar-40- Ar-39 dating on hydrothermal adularia crystals from vugs in the dacite breccia in the Cetate open pit. Microthermometric measurements of fluid inclusions in quartz phenocrysts from the Montana dacite revealed two fluid types that are absent from the hydrothermal breccia and must have been trapped at depth prior to dacite dome emplacement: brine inclusions (32-55 -wt % NaCl equiv, homogenizing at T-h > 460 degrees C) and intermediate density fluids (4.9-15.6 wt % NaCl equiv, T, between 345 degrees-430 degrees C). Secondary aqueous fluid inclusion assemblages in the phenocrysts have salinities of 0.2 to 2.2 wt percent NaCl equiv and T-h of 200 degrees to 280 degrees C. Fluid inclusion assemblages in hydrothermal quartz from breccias and veins have salinities of 0.2 to 3.4 wt percent NaCl equiv and T-h, from 200 degrees to 270 degrees C. The oxygen isotope composition of several zones of an ore-related epithermal quartz crystal indicate a very constant delta O-18 of 4.5 to 5.0 per mil for the mineralizing fluid, despite significant salinity and temperature variation over time. Following microthermometry, selected fluid inclusion assemblages were analyzed by laser ablation-inductively coupled-plasma mass spectrometry (LA-ICMS). Despite systematic differences in salinity between phenocryst-hosted fluids trapped at depth and fluids from quartz in the epithermal breccias, all fluids have overlapping major and trace cation ratios, including identical Na/K/Rb/Sr/Cs/Ba. Consistent with the constant near-magmatic oxygen isotope composition of the hydrothermal fluids, these data strongly indicate a common magmatic component of these chemically conservative solutes in all fluids. Cu, Pb, Zn, and Mn show variations in concentration relative to the relatively non-reactive alkalis, reflecting the precipitation of sulfide minerals together with An in the epithermal breccia, and possibly of Cu in an inferred subjacent porphyry environment. The magmatic-hydrothermal processes responsible for epithermal Au-Ag mineralization at Rosia Montana are, however, not directly related to the formation of the spatially associated porphyry Cu-Au deposit of Rosia Poieni, which occurred lout 3 m.y. later.