Pratap C. Naha

Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells.

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of carboxy H2DCFDA to DCF both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at approximately 4 h), TNF-alpha and IL-6 secretion (maximum at approximately 24 h), MIP-2 levels and cell death (approximately 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.

Nanoparticle Contrast Agents for Computed Tomography: A Focus on Micelles

Computed tomography (CT) is an X-ray-based whole-body imaging technique that is widely used in medicine. Clinically approved contrast agents for CT are iodinated small molecules or barium suspensions. Over the past seven years there has been a great increase in the development of nanoparticles as CT contrast agents. Nanoparticles have several advantages over small molecule CT contrast agents, such as long blood-pool residence times and the potential for cell tracking and targeted imaging applications. Furthermore, there is a need for novel CT contrast agents, owing to the growing population of renally impaired patients and patients hypersensitive to iodinated contrast. Micelles and lipoproteins, a micelle-related class of nanoparticle, have notably been adapted as CT contrast agents. In this review we discuss the principles of CT image formation and the generation of CT contrast. We discuss the progress in developing nontargeted, targeted and cell tracking nanoparticle CT contrast agents. We feature agents based on micelles and used in conjunction with spectral CT. The large contrast agent doses needed will necessitate careful toxicology studies prior to clinical translation. However, the field has seen tremendous advances in the past decade and we expect many more advances to come in the next decade.

Intracellular localisation, geno- and cytotoxic response of polyN-isopropylacrylamide (PNIPAM) nanoparticles to human keratinocyte (HaCaT) and colon cells (SW 480)

PNIPAM nanoparticles, with and without a covalently linked fluorescent label, were prepared by a free radical polymerisation technique. The cyto- and genotoxicity of PNIPAM nanoparticles were analysed in two representative mammalian cell lines, SW480, a colon, and HaCaT, a dermal cell line. Physical characterisation in terms of particle size and zeta potential of the PNIPAM nanoparticles was carried out both in aqueous solution and in the appropriate cell culture media. Uptake and co-localisation of fluorescently labelled PNIPAM nanoparticles was monitored in both cell lines using confocal laser scanning microscope. Genotoxicity analysis using the Comet assay was performed in both cell lines to evaluate any DNA damage. It was observed that the PNIPAM nanoparticles were internalized and localised in lysosomes within 24h. No significant cytotoxic response (p<or.05) was observed in either cell line over concentration ranges from 25 to 1000mg/l for all exposure time periods. Furthermore, no significant genotoxic response (p<or.05) was observed in either cell line over concentration ranges from 12.5 to 800mg/l for all exposure time periods. The results suggest that the PNIPAM nanoparticles show excellent biocompatibility in vitro.

Preparation, characterization of NIPAM and NIPAM/BAM copolymer nanoparticles and their acute toxicity testing using an aquatic test battery

Poly N-isopropylacrylamide and N-isopropylacrylamide/N-tert-butylacrylamide copolymer nanoparticles of 50-70 nm were prepared by free radical polymerisation. The particle sizes of the copolymer nanoparticles were measured in the test media Milli-Q water, Algae Media, Daphnia Media and Microtox Diluent as a function of temperature. Whereas in Milli-Q water the particle size was seen to decrease above the lower critical solution temperature of the thermoresponsive polymer, in the test media it was seen to increase significantly, indicative of aggregation. At the temperatures employed for the ecotoxicological studies all particles, with the exception of the 50:50 copolymer existed as nanoparticles. The zeta potential of Poly N-isopropylacrylamide and N-isopropylacrylamide/N-tert-butylacrylamide copolymer particles measured in the different media was seen to correlate well with the ratio of N-tert-butylacrylamide monomer and therefore the hydrophobicity of the particles. Ecotoxicological studies of the copolymer nanoparticles was performed using four test species Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna, Thamnocephalus platyurus and the cytotoxicity of the 100% Poly N-isopropylacrylamide and 85:15 N-isopropylacrylamide/N-tert-butylacrylamide copolymer nanoparticles was evaluated using a salmonid cell line. Although no significant cytotoxicological response was recorded, significant ecotoxicological response was observed at particle concentrations of up to 1000 mg l(-1). The ecotoxicological response was seen to correlate well with the ratio of N-tert-butylacrylamide monomer and therefore with the zeta potential of the nanoparticles. The toxic response in Daphnia magna was seen to further correlate with the reduction in zeta potential pointing towards a contribution of secondary effects due to modification of the medium. No correlation with particle size was observed. The sensitivity of the test species was seen to vary depending on copolymer composition. The relevance of the derived structure-activity relationships is discussed.

Dextran coated bismuth–iron oxide nanohybrid contrast agents for computed tomography and magnetic resonance imaging

Bismuth nanoparticles have been proposed as a novel CT contrast agent, however few syntheses of biocompatible bismuth nanoparticles have been achieved. We herein report the synthesis of composite bismuth-iron oxide nanoparticles (BION) that are based on a clinically approved, dextran-coated iron oxide formulation; the particles have the advantage of acting as contrast agents for both CT and MRI. BION were synthesized and characterized using various analytical methods. BION CT phantom images revealed that the X-ray attenuation of the different formulations was dependent upon the amount of bismuth present in the nanoparticle, while T2-weighted MRI contrast decreased with increasing bismuth content. No cytotoxicity was observed in Hep G2 and BJ5ta cells after 24 hours incubation with BION. The above properties, as well as the yield of synthesis and bismuth inclusion efficiency, led us to select the Bi-30 formulation for in vivo experiments, performed in mice using a micro-CT and a 9.4 T MRI system. X-ray contrast was observed in the heart and blood vessels over a 2 hour period, indicating that Bi-30 has a prolonged circulation half-life. Considerable signal loss in T2-weighted MR images was observed in the liver compared to pre-injection scans. Evaluation of the biodistribution of Bi-30 revealed that bismuth is excreted via the urine, with significant concentrations found in the kidneys and urine. In vitro experiments confirmed the degradability of Bi-30. In summary, dextran coated BION are biocompatible, biodegradable, possess strong X-ray attenuation properties and also can be used as T2-weighted MR contrast agents.

Synthesis, X-ray Opacity, and Biological Compatibility of Ultra-High Payload Elemental Bismuth Nanoparticle X-ray Contrast Agents

Inorganic nanoscale X-ray contrast agents (XCAs) offer many potential advantages over currently used intravascular molecular contrast agents, including longer circulation and retention times, lower administration volumes, and greater potential for site directed imaging. Elemental bismuth nanoparticles (BiNPs) are particularly attractive candidate XCAs due to the low cost, the high atomic number and high density of bismuth, and the likelihood that BiNPs will oxidatively decompose to biocompatible bismuth(III) ions at controlled rates for renal excretion. Herein we describe the synthesis of ultrahigh payload BiNPs in 1,2-propanediol using a borane reducing agent and glucose as a biocompatible surface stabilizer. Both synthetic solvent (1,2-propanediol) and surfactant (glucose) are evident on the BiNP surfaces when analyzed by 1H NMR and FT-IR spectroscopies. These particles contain ∼6 million Bi atoms per NP and have large inorganic cores (74 nm by TEM) compared to their hydrodynamic size (86 nm by DLS). Thus, the dense BiNP core constitutes the majority (∼60%) of each particle’s volume, a necessary property to realize the full potential of nanoscale XCAs. Using quantitative computed tomography in phantom and in vitro imaging studies, we demonstrate that these BiNPs have greater X-ray opacity than clinical small molecule iodinated contrast agents at the same concentrations. We furthermore demonstrate a favorable biocompatibility profile for these BiNPs in vitro. Altogether, these studies indicate that these ultrahigh payload BiNPs, synthesized from known biocompatible components, have promising physical and cytotoxicological properties for use as XCAs.

Generation of intracellular reactive oxygen species and genotoxicity effect to exposure of nanosized polyamidoamine (PAMAM) dendrimers in PLHC-1 cells in vitro

Polyamidoamine (PAMAM) dendrimers have previously been demonstrated to elicit systematically variable cyto- and eco-toxic responses, promising as the basis for structure-activity relationships governing nanotoxicological responses. In this study, increased production of intracellular reactive oxygen species (ROS), genotoxicity and apoptosis due to in vitro exposure of fish hepatocellular carcinoma cells to dendrimer generations G4, G5 and G6 is demonstrated. A PAMAM dendrimer generation dependent increase in ROS and genotoxicity was observed, consistent with our previous studies. The toxicological responses correlate well with the nanoparticle surface chemistry, specifically, the number of surface amino groups per generation. Although ROS production initially increases approximately linearly, it saturates at higher doses. Notably, normalized to the molar dose of surface amino groups, the dose-dependent ROS production for different generations overlap exactly, indicating that the response is due to these functional units. The genotoxicity response is also well correlated to the number of surface amino groups and therefore generation of PAMAM dendrimers. The observed genotoxicity, related to DNA damage, is related to the generation and dose dependent production of intracellular ROS, at low levels. At the higher ROS levels, increased DNA damage is associated with the onset of necrosis.

Reactive Oxygen Species Mediated DNA Damage In Human Lung Alveolar Epithelial (A549) Cells From Exposure To Non-Cytotoxic MFI-Type Zeolite Nanoparticles

Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50nm and 100nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions. Live cell imaging showed that the nanoparticles precipitated from the colloidal suspension of cell culture media as large agglomerates, coming in contact with the cell surface through sedimentation. A cellular proliferative capacity test showed the zeolite nanoparticles to exhibit no significant cytotoxicity below a concentration of 100μg/ml. However, both the MFI-50 and MFI-100 nanoparticles induced high intracellular reactive oxygen species (ROS) generation and elevated mitochondrial membrane potential in the A549 cells over the measured time period of 12h and at concentrations up to ≤50μg/ml. DNA fragmentation analysis using the comet assay showed that the MFI-50 and MFI-100 nanoparticles cause genotoxicity in a concentration dependent manner. Furthermore, the rate at which maximum genomic damage was caused by MFI-100 nanoparticles in the A549 cells was found to be high as compared to the MFI-50 nanoparticles. However, the damage caused by the MFI-50 nanoparticles was found to accumulate over a longer period of time as compared to MFI-100 nanoparticles. The study therefore points towards the capability of the non-cytotoxic zeolite nanoparticles to induce oxidative stress resulting in short-term altered cellular metabolism up-regulation and genomic instability. Although the damage was found to be short-lived, its persistence over longer durations, or stabilization cannot be neglected. Further studies are in progress to yield a better understanding of the mechanisms for oxidative stress and resulting cascade of events leading to genetic damage in the human lung alveolar epithelial cells following exposure to zeolite nanoparticles of different sizes.

Lipoproteins and lipoprotein mimetics for imaging and drug delivery.

Lipoproteins are a set of natural nanoparticles whose main role is the transport of fats within the body. While much work has been done to develop synthetic nanocarriers to deliver drugs or contrast media, natural nanoparticles such as lipoproteins represent appealing alternatives. Lipoproteins are biocompatible, biodegradable, non-immunogenic and are naturally targeted to some disease sites. Lipoproteins can be modified to act as contrast agents in many ways, such as by insertion of gold cores to provide contrast for computed tomography. They can be loaded with drugs, nucleic acids, photosensitizers or boron to act as therapeutics. Attachment of ligands can re-route lipoproteins to new targets. These attributes render lipoproteins attractive and versatile delivery vehicles. In this review we will provide background on lipoproteins, then survey their roles as contrast agents, in drug and nucleic acid delivery, as well as in photodynamic therapy and boron neutron capture therapy.

Systematic in vitro toxicological screening of gold nanoparticles designed for nanomedicine applications.

Gold nanoparticles (AuNP) are increasingly being applied in the biomedical field as therapeutics, contrast agents, and in diagnostic systems, motivating investigations of their toxicity that might arise from accidental exposure. While other work has investigated the toxicological response to gold nanoparticles for industrial purposes, here we have surveyed formulations that have been developed for biomedical use, are in clinical trials or have been FDA-approved. The AuNP library tested contains a range of shapes, including spheres, rods and shells, that possess a range of coatings, such as silica, citrate, lipoprotein, polymaleic acid, polyethylene glycol, DNA and others. Good cytocompatibility for all formulations was observed after 1 h of incubation. However after 24 h exposure, a nanorod and a spherical DNA coated formulation resulted in toxicity. The coating material was the only factor that influenced toxicity. AuNP exposure seemed to have no effect on cell cytoskeleton deformation and cell spreading. Cell uptake, as measured by computed tomography and ICP-OES, as well as TEM images of cells, confirmed strong AuNP uptake for certain formulations, but there was no correlation with toxicity. No glove translocation occurred, therefore, nitrile gloves are an adequate safety precaution for working with the AuNP studied. In conclusion, the majority of AuNP formulations tested have very low adverse effects.