Scatter Enhanced Phase Contrast Microscopy for Discriminating Mechanisms of Active Nanoparticle Transport in Living Cells.

Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels, which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, which predicts that individual particles down to ∼35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane-assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO2 nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.

Mussel-inspired 3D fiber scaffolds for heart-on-a-chip toxicity studies of engineered nanomaterials.

Due to the unique physicochemical properties exhibited by materials with nanoscale dimensions, there is currently a continuous increase in the number of engineered nanomaterials (ENMs) used in consumer goods. However, several reports associate ENM exposure to negative health outcomes such as cardiovascular diseases. Therefore, understanding the pathological consequences of ENM exposure represents an important challenge, requiring model systems that can provide mechanistic insights across different levels of ENM-based toxicity. To achieve this, we developed a mussel-inspired 3D microphysiological system (MPS) to measure cardiac contractility in the presence of ENMs. While multiple cardiac MPS have been reported as alternatives to in vivo testing, most systems only partially recapitulate the native extracellular matrix (ECM) structure. Here, we show how adhesive and aligned polydopamine (PDA)/polycaprolactone (PCL) nanofiber can be used to emulate the 3D native ECM environment of the myocardium. Such nanofiber scaffolds can support the formation of anisotropic and contractile muscular tissues. By integrating these fibers in a cardiac MPS, we assessed the effects of TiO2 and Ag nanoparticles on the contractile function of cardiac tissues. We found that these ENMs decrease the contractile function of cardiac tissues through structural damage to tissue architecture. Furthermore, the MPS with embedded sensors herein presents a way to non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. These results demonstrate the utility of our MPS as an analytical platform for understanding the functional impacts of ENMs while providing a biomimetic microenvironment to in vitro cardiac tissue samples. Graphical Abstract Heart-on-a-chip integrated with mussel-inspired fiber scaffolds for a high-throughput toxicological assessment of engineered nanomaterials.

Gadolinium-doped silica nanoparticles encapsulating indocyanine green for near infrared and magnetic resonance imaging.

Clinical applications of the indocyanine green (ICG) dye, the only near infrared (NIR) imaging dye approved by the Food and Drug Administration (FDA) in the USA, are limited due to rapid protein binding, fast clearance, and instability in physiologically relevant conditions. Encapsulating ICG in silica particles can enhance its photostability, minimize photobleaching, increase the signal-to-noise (S/N) ratio and enable in vivo studies. Furthermore, a combined magnetic resonance (MR) and NIR imaging particulate can integrate the advantage of high-resolution 3D anatomical imaging with high-sensitivity deep-tissue in-vivo fluorescent imaging. In this report, a novel synthesis technique that can achieve these goals is presented. A reverse-microemulsion-based synthesis protocol is employed to produce 25 nm ICG-doped silica nanoparticles (NPs). The encapsulation of ICG is achieved by manipulating coulombic attractions with bivalent ions and aminated silanes and carrying out silica synthesis in salt-catalyzed, mildly basic pH conditions using dioctyl sulfosuccinate (AOT)/heptane/water microemulsion system. Furthermore, paramagnetic properties are imparted by chelating paramagnetic Gd to the ICG-doped silica NPs. Aqueous ICG-dye-doped silica NPs show increased photostability (over a week) and minimal photobleaching as compared to the dye alone. The MR and optical imaging capabilities of these particles are demonstrated through phantom, in vitro and in vivo experiments. The described particles have the potential to act as theranostic agents by combining photodynamic therapy through the absorption of NIR irradiated light.

The promise of nanotechnology for solving clinical problems in breast cancer.

Approaches for breast cancer treatment are invasive, disfiguring, have significant side-effects, and are not always curative. Nanotechnology is an emerging area which is focused on engineering of materials <100 × 10(-9) m. There is significant promise for advancing nanotechnology to improve breast cancer diagnosis and treatment including non-invasive therapy, monitoring response to therapy, advanced imaging, treatment of metastatic disease, and improved nodal staging. Current approaches and important future directions are discussed.

Nanoparticles as contrast agents for in-vivo bioimaging: current status and future perspectives.

Nanoparticle-based contrast agents are quickly becoming valuable and potentially transformative tools for enhancing medical diagnostics for a wide range of in-vivo imaging modalities. Compared with conventional molecular-scale contrast agents, nanoparticles (NPs) promise improved abilities for in-vivo detection and potentially enhanced targeting efficiencies through longer engineered circulation times, designed clearance pathways, and multimeric binding capacities. However, NP contrast agents are not without issues. Difficulties in minimizing batch-to-batch variations and problems with identifying and characterizing key physicochemical properties that define the in-vivo fate and transport of NPs are significant barriers to the introduction of new NP materials as clinical contrast agents. This manuscript reviews the development and application of nanoparticles and their future potential to advance current and emerging clinical bioimaging techniques. A focus is placed on the application of solid, phase-separated materials, for example metals and metal oxides, and their specific application as contrast agents for in-vivo near-infrared fluorescence (NIRF) imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), ultrasound (US), and photoacoustic imaging (PAI). Clinical and preclinical applications of NPs are identified for a broad spectrum of imaging applications, with commentaries on the future promise of these materials. Emerging technologies, for example multifunctional and theranostic NPs, and their potential for clinical advances are also discussed.

Targeted delivery of amikacin into granuloma.

RATIONALE: Nontuberculous mycobacterial (NTM) infection is a growing problem in the United States and remains underrecognized in the developing world. The management of NTM infections is further complicated by several factors, including the need to use high systemic doses of toxic agents, the length of therapy, and the development of drug resistance. OBJECTIVES: We have evaluated the use of monocyte-derived dendritic cells (DCs) as a delivery vehicle for a luminescent derivative of amikacin prepared by conjugation to fluorescein isothiocyanate (FITC) (amikacin-FITC) into granulomas formed in the tissues of mice infected with Mycobacterium avium. METHODS: Amikacin-FITC was prepared and quantitative fluorescence was used to track the intracellular uptake of this modified antibiotic. The antibiotic activity of amikacin-FITC was also determined to be comparable to unmodified amikacin against M. avium. Amikacin-FITC-loaded DCs were first primed with M. avium, and then the cells were injected into the tail vein of infected mice. After 24 hours, the mice were sacrificed and the tissues were analyzed under fluorescence microscope. MEASUREMENTS AND MAIN RESULTS: We found that we were able to deliver amikacin into granulomas in a mouse model of disseminated mycobacterial infection. No increase in levels of monocyte chemoattractant protein-1 and its CCR2 as markers of inflammation were found when DCs were treated with amikacin-FITC. CONCLUSIONS: DC-based drug delivery may be an adjunct and useful method of delivering high local concentrations of antibiotics into mycobacterial granulomas.

The 'Gator' Mouse Suit for early bioluminescent metastatic breast cancer detection and nanomaterial signal enhancement during live animal imaging.

UNLABELLED: Optical imaging is a cornerstone of modern oncologic research. The aim of this study is to determine the value of a new tool to enhance bioluminescent and fluorescent sensitivity for facilitating very-low-level signal detection in vivo. EXPERIMENTAL: For bioluminescent imaging experiments, a luciferase expressing breast cancer cell line with metastatic phenotype was implanted orthotopically into the mammary fat pad of mice. For fluorescent imaging experiments, near-infrared (NIR) nanoparticles were injected intratumorally and subcutaneously into mice. Images were compared in mice with and without application of the 'Gator' Mouse Suit (GMS). RESULTS: The GMS was associated with early detection and quantification of metastatic bioluminescent very-low-level signal not possible with conventional imaging strategies. Similarly, NIR nanoparticles that were undetectable in locations beyond the primary injection site could be visualized and their very-low-level signal quantifiable with the aid of the GMS. CONCLUSION: The GMS is a device which has tremendous potential for facilitating the development of bioluminescent models and fluorescent nanomaterials for translational oncologic applications.

Copper coated silica nanoparticles for odor removal.

Copper species coated silica nanoparticles (CuOXS) were synthesized for odor removal application. Coating with copper increased the capacity of silica nanoparticles for eliminating a model odor-ethyl mercaptan. Surface area, pore size distribution, and electron paramagnetic resonance spectroscopy analyses indicated that, at lower copper concentrations, copper species preferentially adsorb in 20 Å pores of silica. These copper species in a dispersed state are effective in catalytic removal of ethyl mercaptan. The best performance of copper-coated silica nanoparticles was achieved at a copper concentration of 3 wt %, at which all 20 Å nanopores were filled with isolated copper species. At higher copper loading, copper species are present as clusters on silica surfaces, which were found to be less effective in removing ethyl mercaptan. Gas chromatography experiments were carried out to verify catalytic conversion of ethyl mercaptan to diethyl disulfide by CuOXS particles. The present study suggests that the nature of the copper species and their site of adsorption, as well as state of dispersion, are important parameters to be considered for catalytic removal of sulfur-containing compounds. These parameters are critical for designing high-performance catalytic copper-coated silica nanoparticles for applications such as deodorization, removal of sulfur compounds from crude oil, hydrogenation, and antimicrobial activity.

Synthesis of Precision Gold Nanoparticles Using Turkevich Method.

Gold nanoparticles (AuNPs) exhibit unique size-dependent physiochemical properties that make them attractive for a wide range of applications. However, the large-scale availability of precision AuNPs has been minimal. Not only must the required nanoparticles be of precise size and morphology, but they must also be of exceedingly narrow size distribution to yield accurate and reliable performance. The present study aims to synthesize precision AuNPs and to assess the advantages and limitations of the Turkevich method-one of the common chemical synthesis technique. Colloidal AuNPs from 15 nm to 50 nm in diameter were synthesized using the Turkevich method. The effect of the molar ratio of the reagent mixture (trisodium citrate to gold chloride), the scaled-up batch size, the initial gold chloride concentration, and the reaction temperature was studied. The morphology, optical property, surface chemistry, and chemical composition of AuNPs were thoroughly characterized. It was determined that the as-synthesized AuNPs between 15 nm and 30 nm exhibit well-defined size and shape, and narrow size distribution (PDI < 0.20). However, the AuNPs became more polydispersed and less spherical in shape as the particle size increased.

Effect of UVA irradiance on photocatalytic and UVA inactivation of Bacillus cereus spores.

The current study is the first to delineate the contribution of photocatalysis to inactivation of Bacillus cereus endospores on dry surfaces over a broad range (0-153W/m(2)) of UVA irradiance. Inactivation of spores at low UVA irradiance (30W/m(2)) was primarily due to photocatalysis, whereas at higher UVA irradiance inactivation was primarily due to UV alone. A linear relationship between UVA irradiance and the rate of spore inactivation was observed in the absence of photocatalyst. The rate of photocatalytic inactivation was non-linear with respect to UVA irradiance, exhibiting a maximum at 30W/m(2).

Particle assisted removal of microbes from surfaces.

Increased reliance on kill based approaches for disinfection raises concerns of antimicrobial resistance development and has significantly elevated the need for alternate approaches for skin and substrate disinfection. This study focuses on reducing harmful microbes from substrates primarily via removal and to a lesser extent by kill. HYPOTHESIS: Functional micro-particles designed to adhere to microbes, with a force greater than the force of microbial adhesion to the substrate, would result in enhanced removal-based disinfection of substrates when subject to an external force. EXPERIMENTS: Silica particles were functionalized with a cationic polymer to bind strongly with bacteria via Coulombic interactions. Disinfection efficacies of substrates with functional particles and control groups were evaluated under conditions relevant for handwashing. FINDINGS: Functionalized silica micro-particles result in ∼4 log reduction of E. coli from an artificial skin substrate in 30 s as compared to a maximum of 1.5 log reduction with control particles. Bacterial viability assays indicate a mechanism of action driven by enhanced removal of bacteria with minimal kill. Particle number density, size and suspension velocity along with strong particle - bacteria interactions have been found to be the primary factors responsible for the enhanced bacterial removal from surfaces.

Effect of chain length on binding of fatty acids to Pluronics in microemulsions.

We investigated the effect of fatty acid chain length on the binding capacity of drug and fatty acid to Pluronic F127-based microemulsions. This was accomplished by using turbidity experiments. Pluronic-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated Amitriptyline, an antidepressant drug. Sodium salts of C(8), C(10), or C(12) fatty acid were used in preparation of the microemulsion and the corresponding binding capacities were observed. It has been previously determined that, for microemulsions prepared with sodium caprylate (C(8) fatty acid soap), a maximum of 11 fatty acid molecules bind to the microemulsion per 1 molecule of Pluronic F127 and a maximum of 12 molecules of Amitriptyline bind per molecule of F127. We have found that with increasing the chain length of the fatty acid salt component of the microemulsion, the binding capacity of both the fatty acid and the Amitriptyline to the microemulsion decreases. For sodium salts of C(8), C(10) and C(12) fatty acids, respectively, a maximum of approximately 11, 8.4 and 8.3 molecules of fatty acid molecules bind to 1 Pluronic F127 molecule. We propose that this is due to the decreasing number of free monomers with increasing chain length. As chain length increases, the critical micelle concentration (cmc) decreases, thus leading to fewer monomers. Pluronics are symmetric tri-block copolymers consisting of propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (PEO) blocks. The PEO blocks are hydrophilic while PPO is hydrophobic portion in the Pluronic molecule. Due to this structure, we propose that the fatty acid molecules that are in monomeric form most effectively diffuse between the PEO "tails" and bind to the hydrophobic PPO groups.

Contaminant-Activated Visible Light Photocatalysis.

Pristine titanium dioxide (TiO2) absorbs ultraviolet light and reflects the entire visible spectrum. This optical response of TiO2 has found widespread application as white pigments in paper, paints, pharmaceuticals, foods and plastic industries; and as a UV absorber in cosmetics and photocatalysis. However, pristine TiO2 is considered to be inert under visible light for these applications. Here we show for the first time that a bacterial contaminant (Staphylococcus aureus-a MRSA surrogate) in contact with TiO2 activates its own photocatalytic degradation under visible light. The present study delineates the critical role of visible light absorption by contaminants and electronic interactions with anatase in photocatalytic degradation using two azo dyes (Mordant Orange and Procion Red) that are highly stable because of their aromaticity. An auxiliary light harvester, polyhydroxy fullerenes, was successfully used to accelerate photocatalytic degradation of contaminants. We designed a contaminant-activated, transparent, photocatalytic coating for common indoor surfaces and conducted a 12-month study that proved the efficacy of the coating in killing bacteria and holding bacterial concentrations generally below the benign threshold. Data collected in parallel with this study showed a substantial reduction in the incidence of infections.

Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials.

The possible combination of specific physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. We have investigated the importance of proteins adsorbed onto the surface of two distinct classes of nanomaterials (single-walled carbon nanotubes [SWCNTs]; 10-nm amorphous silica) in guiding nanomaterial uptake or toxicity in the RAW 264.7 macrophage-like model. Albumin was identified as the major fetal bovine or human serum/plasma protein adsorbed onto SWCNTs, while a distinct protein adsorption profile was observed when plasma from the Nagase analbuminemic rat was used. Damaged or structurally altered albumin is rapidly cleared from systemic circulation by scavenger receptors. We observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 h) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist). Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647). Precoating SWCNTs with a nonionic surfactant (Pluronic F127) inhibited albumin adsorption and anti-inflammatory properties. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction under serum-free conditions. SWCNTs did not reduce binding of LPS(Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica particles (50-1000 nm) was qualitatively different, relative to SWCNTs, and precoating amorphous silica with Pluronic F127 dramatically reduced the adsorption of serum proteins and toxicity. Collectively, these observations suggest an important role for adsorbed proteins in modulating the uptake and toxicity of SWCNTs and nano-sized amorphous silica.

EphA2 targeted intratumoral therapy for non-small cell lung cancer using albumin mesospheres.

Lung cancer, primarily non-small cell lung cancer (NSCLC), is the leading cause of cancer mortality and the prognosis of patients with advanced or metastatic NSCLC is poor. Despite significant advances in diagnosis and treatment, little improvement has been seen in NSCLC mortality. Recently, Intratumoral Chemotherapy, a direct local delivery of chemotherapeutic drugs, has shown promise in clinical studies. However, toxicity and high dosage of chemotherapeutic agents used for treatment are a limitation. Moreover, these drugs damage indiscriminately, cancerous as well as normal tissues. Thus, a novel therapeutic strategy that targets only malignant tissue sparing normal tissue becomes an urgent issue. Ephrin receptor-A2 (EphA2), a new biomarker, is over-expressed in NSCLC, but not on normal epithelial cells. Receptor EphA2 is a cell surface protein, which upon binding to its ligand EphrinA1 undergo phosphorylation and degradation which attenuates NSCLC growth. Targeting the tumor, sparing the normal tissue and enhancing the therapeutic effects of ligand proteins are the goal of this project. Thus a novel method, intratumoral EphA2 targeted therapy, has been developed to target the oncogenic receptors on tumor tissue by using albumin mesosphere (AMS) conjugated ephrinA1 in mice bearing NSCLC tumors.

Research strategies for safety evaluation of nanomaterials, part V: role of dissolution in biological fate and effects of nanoscale particles.

Dissolution, translocation, and disposition have been shown to play a key role in the fate and effects of inhaled particles and fibers. Concepts that have been applied in the micron size range may be usefully applied to the nanoscale range, but new challenges are presented based on the small size and possible change in the dissolution:translocation relationship. The size of the component molecule itself may be on the nanoscale. Solute concentration, surface area, surface morphology, surface energy, dissolution layer properties, adsorbing species, and aggregation are relevant parameters in considering dissolution at the nanoscale. With regard to the etiopathology caused by these types of particulates, the metrics of dose (particle number, surface area, mass or shape) is not yet well defined. Analytical procedures for assessing dissolution and translocation include chemical assay and particle characterization. Leaching of substituents from particle surfaces may also be important. Compartmentalization within the respiratory tract may add another dimension of complexity. Dissolution may be a critical step for some nanoscale materials in determining fate in the environment and within the body. This review, combining aspects of particle toxicology, material science, and analytical chemistry, is intended to provide a useful basis for developing relevant dissolution assay(s) for nanoscale particles.

Research strategies for safety evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation.

To properly assign mechanisms or causes for toxic effects of nanoscale materials, their properties and characteristics both outside and within the biological environment must be well understood. Scientists have many tools for studying the size, shape, and surface properties of particulates outside of the physiological environment; however, it is difficult to measure many of these same properties in situ without perturbing the environment, leading to spurious findings. Characterizing nanoparticle systems in situ can be further complicated by an organism's active clearance, defense, and/or immune responses. As toxicologists begin to examine nanomaterials in a systematic fashion, there is consensus that a series of guidelines or recommended practices is necessary for basic characterization of nanomaterials. These recommended practices should be developed jointly by physical scientists skilled in nano characterization and biological scientists experienced in toxicology research. In this article, basic nanoparticle characterization techniques are discussed, along with the some of the issues and implications associated with measuring nanoparticle properties and their interactions with biological systems. Recommendations regarding how best to approach nanomaterial characterization include using proper sampling and measurement techniques, forming multidisciplinary teams, and making measurements as close to the biological action point as possible.

Nanoparticles for bioimaging.

The emergence of synthesis strategies for the fabrication of nanosized contrast agents is anticipated to lead to advancements in understanding biological processes at the molecular level in addition to progress in the development of diagnostic tools and innovative therapies. Imaging agents such as fluorescent dye-doped silica nanoparticles, quantum dots and gold nanoparticles have overcome many of the limitations of conventional contrast agents (organic dyes) such as poor photostability, low quantum yield, insufficient in vitro and in vivo stability, etc. Such particulates are now being developed for absorbance and emission in the near infrared region, which is expected to allow for real time and deep tissue imaging via optical routes. Other efforts to facilitate deep tissue imaging with pre-existing technologies have lead to the development of multimodal nanoparticles which are both optical and MRI active. The main focus of this article is to provide an overview of properties and design of contrast agents such as dye-doped silica nanoparticles, quantum dots and gold nanoparticles for non-invasive bioimaging.

Enhancement of titanium dioxide photocatalysis by water-soluble fullerenes.

Fullerenes are known for their unique electronic properties including high electron affinity. Although use of fullerenes for scavenging photo-generated electrons from titanium dioxide particles has been demonstrated, no attempts have been made to utilize the unique properties of fullerenes to increase the efficacy of photocatalysis. The present study has demonstrated that a mixture of water-soluble polyhydroxy fullerenes (PHF) and titanium dioxide (anatase polymorph) enhances photocatalytic degradation of organic dye. The PHF molecules adsorbed to the surface of titanium dioxide due to electrostatic forces, with adsorption density being higher at lower pH values. The surface coverage of titanium dioxide nanoparticles by PHF molecules determined the extent of enhancement, with an optimum dosed weight ratio of PHF to titanium dioxide at 0.001. Hydroxylation and concomitant solubilization of fullerenes allow their unique electronic properties to be harnessed for photocatalysis.