Facile biosynthesis, separation and conjugation of gold nanoparticles to doxorubicin

Particle shape and size determine the physicochemical and optoelectronic properties of nanoscale materials, including optical absorption, fluorescence, and electric and magnetic moments. It is thus desirable to be able to synthesize and separate various particle shapes and sizes. Biosynthesis using microorganisms has emerged as a more ecologically friendly, simpler, and more reproducible alternative to chemical synthesis of metal and semiconductor nanoparticles, allowing the generation of rare forms such as triangles. Here we show that the plant pathogenic fungus Helminthosporum solani, when incubated with an aqueous solution of chloroaurate ions, produces a diverse mixture of extracellular gold nanocrystals in the size range from 2 to 70?nm. A plurality are polydisperse spheres, but a significant number are homogeneously sized rods, triangles, pentagons, pyramids, and stars. The particles can be separated according to their size and shape by using a sucrose density gradient in a tabletop microcentrifuge, a novel and facile approach to nanocrystal purification. Conjugation to biomolecules can be performed without further processing, as illustrated with the smallest fraction of particles which were conjugated to the anti-cancer drug doxorubicin (Dox) and taken up readily into HEK293 cells. The cytotoxicity of the conjugates was comparable to that of an equivalent concentration of Dox.     

Surface modification of gamma-Fe2O3@SiO2 magnetic nanoparticles for the controlled interaction with biomolecules

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.     

A method for determination of particle magnetic susceptibility with analytical magnetapheresis

We recently developed a new method for simple determination of particle magnetic susceptibility using analytical magnetapheresis. This new method does not require laborious calibration plots and trial susceptibility values as do previous analytical magnetapheresis methods. The new method is based on balancing channel flow rates and magnetically induced flow rates for particle deposition in analytical magnetapheresis. The maximal flow rate for complete particle deposition was determined experimentally and set to equal the magnetically induced flow rate for determining particle magnetic susceptibility. This magnetic susceptibility determination generally takes less than 20 min. Several magnetically susceptible and ion-labeled particles were tested using this new method. The carrier magnetic susceptibilities were varied, and erbium ion-labeled particles were studied experimentally, resulting in successful susceptibility determinations of erbium ion-labeled particles and yeasts. The precision of each measurement was generally approximately 10%. Experimental determination of particle magnetic susceptibilities differed by less than 10% from reference measurements taken using a superconducting quantum interference device magnetometer. This method can determine minimal susceptibilities on the order of 10(-9) cgs. The minimum number of erbium labeling ions per particle required for complete deposition of silicas and yeasts was found to be 6.7 x 10(9). Analytical magnetapheresis shows good potential for use in simple determination of particle magnetic susceptibilities and should become a useful technique.     

Surface modified superparamagnetic nanoparticles for drug delivery: Interaction studies with human fibroblasts in culture

The concept of drug delivery using magnetic nanoparticles greatly benefit from the fact that nanotechnology has developed to a stage that it makes possible not only to produce magnetic nanoparticles in a very narrow size distribution range with superparamagnetic properties but also to engineer particle surfaces to provide site specific delivery of drugs. The size and surface characteristics of the nanoparticles are crucial factors that determine the success of the particles when used in vivo. The aim of this study was to modify the surfaces of the magnetic nanoparticles with PEG to improve the biocompatibility of the nanoparticles by resisting protein adsorption and increasing their intracellular uptake. In this study, the poly(ethyleneglycol) (PEG) modified superparamagnetic iron oxide nanoparticles have been prepared and their influence on human dermal fibroblasts is assessed in terms of cell adhesion/viability, morphology, particle uptake and cytoskeletal organisation studies. Various techniques have been used to determine nanoparticle-cell interactions including light, fluorescence, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The modification of nanoparticle surface induced alterations in cell behaviour distinct from the unmodified particles, suggesting that cell response can be directed via specifically engineered particle surfaces.     

Preparation and characterization of nanoparticles based on histidine–hyaluronic acid conjugates as doxorubicin carriers

Histidine-hyaluronic acid (His-HA) conjugates were synthesized using hyaluronic acid (HA) as a hydrophilic segment and histidine (His) as hydrophobic segment by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) mediated coupling reactions. The structural characteristics of the His-HA conjugates were investigated using (1)H NMR. His-HA nanoparticles (HH-NPs) were prepared based on His-HA conjugates, and the characteristics of HH-NPs were investigated using dynamic light scattering, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and fluorescence spectroscopy. The particles were between 342 and 732 nm in size, depending on the degree of substitution (DS) of the His. TEM and SEM images indicated that the morphology of HH-NPs was spherical in shape. The critical aggregation concentrations of HH-NPs ranged from 0.034 to 0.125 mg/ml, which decreased with an increase in the DS of the His. Images of fluorescence microscopy indicate that HH-NPs were taken up by the cancer cell line (MCF-7), and significantly decreased by competition inhibition of free HA. From the cytotoxicity test, it was found that DOX-loaded HH-NPs exhibited similar dose and time-dependent cytotoxicity against MCF-7 cells with free DOX.     

Imaging and analysis of immobilized particle arrays

An automated imaging system was developed to quantify fluorescence signals from particles immobilized on hydrogel-coated slides. Arrays of submicrometer-diameter particles were printed with up to 600 particles/spot. The slides were read under 20x magnification without cover slips. Software was written to image individual spots and measure the median particle fluorescence in each spot. To locate array spots, an alignment program made use of two fiducial grids of fluorescent reference particles at either end of the slide. Focusing was adjusted locally using spots of reference particles located at the centers of focusing neighborhoods. The response was linear across a two-decade range, and the precision of readings was better than 5% down to approximately 1000 fluors/particle. Exposure times varied with signal intensity, reaching 1 s at the lowest levels of fluorescence. Data demonstrate feasibility for measuring fluorescence from immobilized particle arrays on an automated microscope with accuracy and precision similar to fluorescence measurements of microparticles with a flow cytometer. This work provides automation of imaging and analysis procedures necessary for development of immobilized particle arrays as an analytical platform that combines advantageous features of both planar and suspension arrays.     

Direct deposition of patterned nanocrystalline CVD diamond using an electrostatic self-assembly method with nanodiamond particles

Micron-sized and precise patterns of nanocrystalline CVD diamond were fabricated successfully on substrates using dispersed nanodiamond particles, charge connection by electrostatic self-assembly, and photolithography processes. Nanodiamond particles which had been dispersed using an attritional milling system were attached electrostatically on substrates as nuclei for diamond growth. In this milling process, poly sodium 4-styrene sulfonate (PSS) was added as an anionic dispersion agent to produce the PSS/nanodiamond conjugates. Ultra dispersed nanodiamond particles with a ζ-potential and average particle size of - 60.5 mV and ～ 15 nm, respectively, were obtained after this milling process. These PSS/nanodiamond conjugates were attached electrostatically to a cationic polyethyleneimine (PEI) coated surface on to which a photoresist had been patterned in an aqueous solution of the PSS/nanodiamond conjugated suspension. A selectively seeded area was formed successfully using the above process. A hot filament chemical vapor deposition system was used to synthesize the nanocrystalline CVD diamond on the seeded area. Micron-sized, thin and precise nanocrystalline CVD diamond patterns with a high nucleation density (3.8 ± 0.4 × 10(11) cm(-2)) and smooth surface were consequently fabricated.     

Real-time observation of Brownian motion and cluster movement of ferro- and non-magnetic particles in magnetic fluids

Cluster movements of ferro- and non-magnetic particles in magnetic fluids were investigated using optical microscope system and image processing system. Real-time visualizations of the Brownian motion of particles and the chain-like cluster movement of both types of particle were performed under a magnetic field. The principal objectives of this study were to clarify the applicability of the optical microscope system and image processing system, and to analyze the growth process of the cluster under magnetic field. The analysis of particle image was done using Particle Tracking Velocimetry (PTV). The results clarified that the real-time observation of Brownian motion and cluster movement of ferro- and non-magnetic particles in magnetic fluids can be carried out using the optical microscope system and the PTV image measurement. Independent continuous measurements with changing positions and velocity of the minute particle were made possible. The study concluded that the system can obtain satisfactory results on growth process measurement of cluster under a magnetic field.     

Real-time observation of Brownian motion and cluster movement of ferro- and non-magnetic particles in magnetic fluids

Cluster movements of ferro- and non-magnetic particles in magnetic fluids were investigated using optical microscope system and image processing system. Real-time visualizations of the Brownian motion of particles and the chain-like cluster movement of both types of particle were performed under a magnetic field. The principal objectives of this study were to clarify the applicability of the optical microscope system and image processing system, and to analyze the growth process of the cluster under magnetic field. The analysis of particle image was done using Particle Tracking Velocimetry (PTV). The results clarified that the real-time observation of Brownian motion and cluster movement of ferro- and non-magnetic particles in magnetic fluids can be carried out using the optical microscope system and the PTV image measurement. Independent continuous measurements with changing positions and velocity of the minute particle were made possible. The study concluded that the system can obtain satisfactory results on growth process measurement of cluster under a magnetic field.     

Study of flow fractionation characteristics of magnetic chromatography utilizing high-temperature superconducting bulk magnet

We present numerical simulation of separating magnetic particles with different magnetic susceptibilities by magnetic chromatography using a high-temperature superconducting bulk magnet. The transient transport is numerically simulated for two kinds of particles having different magnetic susceptibilities. The time evolutions were calculated for the particle concentration in the narrow channel of the spiral arrangement placed in the magnetic field. The field is produced by the highly magnetized high-temperature superconducting bulk magnet. The numerical results show the flow velocity difference of the particle transport corresponding to the difference in the magnetic susceptibility, as well as the possible separation of paramagnetic particles of 20 nm diameter.     

Magnetic multicore nanoparticles for hyperthermia--influence of particle immobilization in tumour tissue on magnetic properties

When using magnetic nanoparticles as a heating source for magnetic particle hyperthermia it is of particular interest to know if the particles are free to move in the interstitial fluid or are fixed to the tumour tissue. The immobilization state determines the relaxation behaviour of the administered particles and thus their specific heating power. To investigate this behaviour, magnetic multicore nanoparticles were injected into experimentally grown tumours in mice and magnetic heating treatment was carried out in an alternating magnetic field (H = 25 kA m(-1), f = 400 kHz). The tested particles were well suited for magnetic heating treatment as they heated a tumour of about 100 mg by about 22 K within the first 60 s. Upon sacrifice, histological tumour examination showed that the particles form spots in the tissue with a mainly homogeneous particle distribution in these spots. The magnetic ex vivo characterization of the removed tumour tissue gave clear evidence for the immobilization of the particles in the tumour tissue because the particles in the tumour showed the same magnetic behaviour as immobilized particles. Therefore, the particles are not able to rotate and a temperature increase due to Brown relaxation can be neglected. To accurately estimate the heating potential of magnetic materials, the respective environments influencing the nanoparticle mobility status have to be taken into account.     

Study of flow fractionation characteristics of magnetic chromatography utilizing high-temperature superconducting bulk magnet

Abstract

We present numerical simulation of separating magnetic particles with different magnetic susceptibilities by magnetic chromatography using a high-temperature superconducting bulk magnet. The transient transport is numerically simulated for two kinds of particles having different magnetic susceptibilities. The time evolutions were calculated for the particle concentration in the narrow channel of the spiral arrangement placed in the magnetic field. The field is produced by the highly magnetized high-temperature superconducting bulk magnet. The numerical results show the flow velocity difference of the particle transport corresponding to the difference in the magnetic susceptibility, as well as the possible separation of paramagnetic particles of 20 nm diameter.     

Fluorescently imaged particle counting immunoassay for sensitive detection of DNA modifications

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.     

Dynamic capture and accumulation of multiple types of magnetic particles based on fully coupled multiphysics model in multiwire matrix for high-gradient magnetic separation

High-gradient magnetic separation (HGMS) effectively separates fine weakly magnetic minerals using a magnetic matrix. The basic principle of single-wire capture of magnetic particles in HGMS has received considerable attention. In practice, however, a real matrix is made of numerous magnetic wires. Transport of magnetic particles inside a multiwire matrix under various operating conditions has not been sufficiently investigated, and it is not clear whether single-wire and multiwire matrices differ significantly. A fully coupled multiphysics model based on the idealized capture model was developed to investigate the 2D capture and accumulation of multiple types of particles in single-wire and multiwire matrices. In this model, the properties of multiple types of particles were defined. Then, particle tracing via the fluid flow model was used to calculate the dynamic capture and accumulation of particles under the determined magnetic and flow fields. The time-dependent dynamic capture mode used in this study can reveal the details of particle capture and accumulation in single-wire and multiwire matrices. All the calculations and analyses indicate that single-wire and multiwire matrices both exhibit basically the similar capture tendency as the particle size, slurry feed velocity, and magnetic induction are gradually increased, and a single-wire matrix always has a much higher capture selectivity than a multiwire matrix. This difference in selectivity between the single-wire and multiwire matrices results mainly from magnetic coupling between magnetic wires in the multiwire matrix, where the fluid flow is also quite complicated. In addition, adjacent columns of wires are staggered vertically, increasing the probability of collisions between the particles and the wires; thus, intergrowth particles that are not captured by the upstream wires are more easily captured by the downstream wires. By comparing the experimental results with the simulation results, the correctness of the HGMS recovery and grade prediction results was verified.     

Control of magnetophoretic mobility by susceptibility-modified solutions as evaluated by cell tracking velocimetry and continuous magnetic sorting

With the analytical expression for the magnetophoretic mobility of an ideal, linearly polarizable sphere undergoing creeping motion in viscous medium, we have shown that both attractive and repulsive motions are possible in the magnetic field. We have validated theoretical predictions using magnetic monodisperse microspheres of 5.2-microm diameter and nonmagnetic polystyrene microspheres of 6.99-microm diameter suspended in solutions of paramagnetic ions. The microsphere magnetophoretic mobility was measured using a modified particle tracking velocimetry system, developed in-house and called a cell tracking velocimeter. The product of measured mobility and viscosity agrees well with the theoretical prediction, differing only by approximately 11%. Further, a 26% increase in resolution between magnetic and nonmagnetic particle distributions was evaluated when paramagnetic ion carrier was used instead of water. Continuous particle sorting based on differences in magnetophoretic mobility was performed with another device developed by us, the quadrupole magnetic flow sorter (QMS). In the QMS, the introduction of paramagnetic ions into the carrier was effective in suppressing nonspecific crossover (i.e., the transport of low-mobility particles into the magnetic particle fraction) in particles and in biologically relevant red blood cells and thus showed promise as a means of increasing the purity of the magnetic separation.     

纳米磁性微球对人血浆抗体的特异性分离过程

采用细乳液聚合法制备了嗜硫磁性纳米微球,磁球粒径较小,具有灵敏的磁响应性。经嗜硫性配基(2-巯基噻唑啉)修饰后,在pH=4.0的低浓度柠檬酸缓冲液中可特异性吸附免疫球蛋白G,并实现了在较低盐浓度下被吸附抗体的有效脱附,在吸附过程中表现出明显的单分子层吸附过程。在分离过程中几乎不损伤抗体的生物活性,所分离抗体的生物活性高于99%。磁性微球的平衡吸附量为53.04mg/g,相比于微米级磁性微球的平衡吸附量(3.1mg/g),提高了17倍。     

A novel experimental technique to determine the heat transfer coefficient between the bed and particles in a downer

A novel method for directly measuring the temperature history of mobile hot ferromagnetic particles (steel particles), substituting for reacting particles, in a binary-solid (reacting particles and inert particles) downflow is introduced. The temperature history of the hot steel particles can be obtained by measuring the temperature of the particles at different axial positions using magnetic fields that can separate the steel particles from other bed materials immediately and easily. Employing the magnetic marking method, magnetic sensors were used to detect the change in magnetic flux density in a given magnetic field, and the residence time of the steel particles was also measured. The cross-sectional averaged particle-to-bed heat transfer coefficients were calculated from the experimental results using simple heat balance equations. The measured temperature data have a relatively wide error range; however, the average temperature curves derived from the average particle-to-bed heat transfer coefficients agreed with the temperature plots. Therefore, the experimental method of this study is applicable to the measurement of the particle temperature in a binary-solid downflow. The results showed that there is strong correlation between the particle-to-bed heat transfer coefficients and normalized collision frequency under the laminar gas flow conditions.     

Multimodal biomedical imaging with asymmetric single-walled carbon nanotube/iron oxide nanoparticle complexes

Magnetic iron oxide nanoparticles and near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWNT) form heterostructured complexes that can be utilized as multimodal bioimaging agents. Fe catalyst-grown SWNT were individually dispersed in aqueous solution via encapsulation by oligonucleotides with the sequence d(GT)15, and enriched using a 0.5 T magnetic array. The resulting nanotube complexes show distinct NIR fluorescence, Raman scattering, and visible/NIR absorbance features, corresponding to the various nanotube species. AFM and cryo-TEM images show DNA-encapsulated complexes composed of a approximately 3 nm particle attached to a carbon nanotube on one end. X-ray diffraction (XRD) and superconducting quantum interference device (SQUID) measurements reveal that the nanoparticles are primarily Fe2O3 and superparamagnetic. The Fe2O3 particle-enriched nanotube solution has a magnetic particle content of approximately 35 wt %, a magnetization saturation of approximately 56 emu/g, and a magnetic relaxation time scale ratio (T1/T2) of approximately 12. These complexes have a longer spin-spin relaxation time (T2 approximately 164 ms) than typical ferromagnetic particles due to the smaller size of their magnetic component while still retaining SWNT optical signatures. Macrophage cells that engulf the DNA-wrapped complexes were imaged using magnetic resonance imaging (MRI) and NIR mapping, demonstrating that these multifunctional nanostructures could potentially be useful in multimodal biomedical imaging.     

Fluorescence‐Encoded Gold Nanoparticles: Library Design and Modulation of Cellular Uptake into Dendritic Cells

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo‐ and hetero‐functional fluorescence‐encoded gold nanoparticles (Au‐NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)‐based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV‐Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence‐activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero‐functionalized Au‐NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.