Enhanced pulsed magneto-motive ultrasound imaging using superparamagnetic nanoclusters

Recently, pulsed magneto-motive ultrasound (pMMUS) imaging augmented with ultra-small magnetic nanoparticles has been introduced as a tool capable of imaging events at molecular and cellular levels. The sensitivity of a pMMUS system depends on several parameters, including the size, geometry and magnetic properties of the nanoparticles. Under the same magnetic field, larger magnetic nanostructures experience a stronger magnetic force and produce larger displacement, thus improving the sensitivity and signal-to-noise ratio (SNR) of pMMUS imaging. Unfortunately, large magnetic iron-oxide nanoparticles are typically ferromagnetic and thus are very difficult to stabilize against colloidal aggregation. In the current study we demonstrate improvement of pMMUS image quality by using large size superparamagnetic nanoclusters characterized by strong magnetization per particle. Water-soluble magnetic nanoclusters of two sizes (15 and 55 nm average size) were synthesized from 3 nm iron precursors in the presence of citrate capping ligand. The size distribution of synthesized nanoclusters and individual nanoparticles was characterized using dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). Tissue mimicking phantoms containing single nanoparticles and two sizes of nanoclusters were imaged using a custom-built pMMUS imaging system. While the magnetic properties of citrate-coated nanoclusters are identical to those of superparamagnetic nanoparticles, the magneto-motive signal detected from nanoclusters is larger, i.e. the same magnetic field produced larger magnetically induced displacement. Therefore, our study demonstrates that clusters of superparamagnetic nanoparticles result in pMMUS images with higher contrast and SNR.     

Labeling TiO2 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of TiO2–DNA Nanoconjugate Stability

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO₂) nanoparticles. These nanoparticles, when electronically linked to single‐stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO₂–DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO₂ nanoparticles and TiO₂–DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO₂ nanoparticle uptake in a large population of living cells (>10⁴ cells). X‐ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO₂–DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO₂–DNA nanoconjugates in malignant cells.     

Preparation and control of the formation of single core and clustered nanoparticles for biomedical applications using a versatile amphiphilic diblock copolymer

We report the application of a versatile diblock copolymer, poly(ethylene oxide)-b-poly(γ-methacryloxypropyl trimethoxysilane) (PEO-b-PγMPS), to prepare nanocrystals such as iron oxide nanoparticles or quantum dots, with either a single core or multi-core cluster, for biomedical applications. This amphiphilic copolymer comprises both a hydrophilic PEO segment and a hydrophobic segment with a “surface anchoring moiety” (the silane group) which can interact effectively with the hydrophobic nanocrystals through ligand exchange. One of the unique features of this work is that we can control the formation of either single core nanoparticles or multi-core nanoclusters by simply varying the conditions of ligand exchange and aging of the mixture of block copolymer and nanoparticles without needing to change the copolymer. The morphologies of the resulting single core nanoparticles or multi-core nanoclusters were confirmed by dynamic light scattering and transmission electron microscopy. The clustered nanoparticles exhibit enhanced physicochemical properties that are beyond those expected from a simple accumulation of individual nanoparticles. Additionally, the hybrid nanoparticles containing both magnetic iron oxide nanoparticles and optical quantum dots obtained using our strategy provide have combined magnetic and optical functionalities that allow for potential new and expanded biomedical applications, as demonstrated by their use for magnetic resonance imaging and biomarker-targeted cell imaging.     

Superparamagnetic iron oxide nanoparticles assembled magnetic nanobubbles and their application for neural stem cells labeling

Micro/nanobubbles for use as ultrasound contrast agents have been fabricated with different shell materials.When various biomedical nanoparticles have been embedded in the shells of bubbles,the composite structures have shown promising applications in multi-modal imaging,drug/gene delivery,and biomedical sensing.In this study,we developed a new gas-liquid interface self-assembly method to prepare magnetic nanobubbles embedded with superparamagnetic iron oxide nanoparticles(SPIONs).The diameter of the generated assembled nanobubbles was 227.40±87.21 nm with a good polydispersity index(PDI)of 0.29.Under the condition of 150 compression cycles,the nanobubble concentration could reach about 6.12×10⁹/mL.Transmission electron microscopy(TEM)and scanning electronic microscopy(SEM)demonstrated that the assembled nanobubbles had a hollow gas core with SPIONs adsorbed on the surface.Ultrasound(US)imaging and magnetic resonance imaging(MRI)experiments indicated that the assembled magnetic nanobubbles exhibited good US and MR contrast capabilities.Moreover,the assembled magnetic nanobubbles were used to label neural stem cells under ultrasound exposure.After 40 s US exposure,the magnetic nanobubbles could be delivered into cells with 2.80 pg Fe per cell,which could be observed in the intracellular endosome by TEM.Compared with common incubation methods,the ultrasound exposure method did not introduce the potential cytotoxicity of transfection reagents and the efficiency was about twice as high as the efficiency of incubation.Therefore,the assembled magnetic nanobubbles prepared through the pressure-driven gas-liquid interface assembly approach could be a potential US/MRI dual model imaging nanocarrier for regenerative applications.     

Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells

Magnetogenetics is emerging as a novel approach for remote‐controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP–nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote‐controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine.     

Three-dimensional reduced-symmetry of colloidal plasmonic nanoparticles

Owing to their novel optical properties, three-dimensional plasmonic nanostructures with reduced symmetry such as a nanocrescent and a nanocup have attracted considerable current interest in biophotonic imaging and sensing. However, their practical applications have been still limited since the colloidal synthesis of such structures that allows, in principle, for in vivo application and large-scale production has not been explored yet. To date, these structures have been fabricated only on two-dimensional substrates using micro/nanofabrication techniques. Here we demonstrate an innovative way of breaking symmetry of colloidal plasmonic nanoparticles. Our strategy exploits the direct overgrowth of Au on a hybrid colloidal dimer consisting of Au and polystyrene (PS) nanoparticles without the self-nucleation of Au in an aqueous solution. Upon the overgrowth reaction, the steric crowding of PS leads to morphological evolution of the Au part in the dimer ranging from half-shell, nanocrescent to nanoshell associated with the appearance of the second plasmon absorption band in near IR. Surface-enhanced Raman scattering signal is obtained directly from the symmetry-broken nanoparticles solution as an example showing the viability of the present approach. We believe our concept represents an important step toward a wide range of biophotonic applications for optical nanoplasmonics such as targeting, sensing/imaging, gene delivery, and optical gene regulations.     

Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer

Gold nanoparticles with unique optical properties may be useful as biosensors in living whole cells. Using a simple and inexpensive technique, we recorded surface plasmon resonance (SPR) scattering images and SPR absorption spectra from both colloidal gold nanoparticles and from gold nanoparticles conjugated to monoclonal anti-epidermal growth factor receptor (anti-EGFR) antibodies after incubation in cell cultures with a nonmalignant epithelial cell line (HaCaT) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). Colloidal gold nanoparticles are found in dispersed and aggregated forms within the cell cytoplasm and provide anatomic labeling information, but their uptake is nonspecific for malignant cells. The anti-EGFR antibody conjugated nanoparticles specifically and homogeneously bind to the surface of the cancer type cells with 600% greater affinity than to the noncancerous cells. This specific and homogeneous binding is found to give a relatively sharper SPR absorption band with a red shifted maximum compared to that observed when added to the noncancerous cells. These results suggest that SPR scattering imaging or SPR absorption spectroscopy generated from antibody conjugated gold nanoparticles can be useful in molecular biosensor techniques for the diagnosis and investigation of oral epithelial living cancer cells in vivo and in vitro.     

FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents

Nanocrystals with advanced magnetic or optical properties have been actively pursued for potential biological applications, including integrated imaging, diagnosis and therapy. Among various magnetic nanocrystals, FeCo has superior magnetic properties, but it has yet to be explored owing to the problems of easy oxidation and potential toxicity. Previously, FeCo nanocrystals with multilayered graphitic carbon, pyrolytic carbon or inert metals have been obtained, but not in the single-shelled, discrete, chemically functionalized and water-soluble forms desired for biological applications. Here, we present a scalable chemical vapour deposition method to synthesize FeCo/single-graphitic-shell nanocrystals that are soluble and stable in water solutions. We explore the multiple functionalities of these core-shell materials by characterizing the magnetic properties of the FeCo core and near-infrared optical absorbance of the single-layered graphitic shell. The nanocrystals exhibit ultra-high saturation magnetization, r1 and r2 relaxivities and high optical absorbance in the near-infrared region. Mesenchymal stem cells are able to internalize these nanoparticles, showing high negative-contrast enhancement in magnetic-resonance imaging (MRI). Preliminary in vivo experiments achieve long-lasting positive-contrast enhancement for vascular MRI in rabbits. These results point to the potential of using these nanocrystals for integrated diagnosis and therapeutic (photothermal-ablation) applications.     

Counting single native biomolecules and intact viruses with color-coded nanoparticles

Nanometer-sized particles such as semiconductor quantum dots and energy-transfer nanoparticles have novel optical properties such as tunable light emission, signal brightness, and multicolor excitation that are not available from traditional organic dyes and fluorescent proteins. Here we report the use of color-coded nanoparticles and dual-color fluorescence coincidence for real-time detection of single native biomolecules and viruses in a microfluidic channel. Using green and red nanoparticles to simultaneously recognize two binding sites on a single target, we demonstrate that individual molecules of genes, proteins, and intact viruses can be detected and identified in complex mixtures without target amplification or probe/target separation. Real-time coincidence analysis of single-photon events allows rapid detection of bound targets and efficient discrimination of excess unbound probes. Quantitative studies indicate that the counting results are remarkably precise when the total numbers of counted molecules are more than 10. The use of bioconjugated nanoparticle probes for single-molecule detection is expected to have important applications in ultrasensitive molecular diagnostics, bioterrorism agent detection, and real-time imaging and tracking of single-molecule processes inside living cells.     

Rapid,High‐Resolution Magnetic Microscopy of Single Magnetic Microbeads

Magnetic microparticles or “beads” are used in a variety of research applications from cell sorting through to optical force traction microscopy. The magnetic properties of such particles can be tailored for specific applications with the uniformity of individual beads critical to their function. However, the majority of magnetic characterization techniques quantify the magnetic properties from large bead ensembles. Developing new magnetic imaging techniques to evaluate and visualize the magnetic fields from single beads will allow detailed insight into the magnetic uniformity, anisotropy, and alignment of magnetic domains. Here, diamond‐based magnetic microscopy is applied to image and characterize individual magnetic beads with varying magnetic and structural properties: ferromagnetic and superparamagnetic/paramagnetic, shell (coated with magnetic material), and solid (magnetic material dispersed in matrix). The single‐bead magnetic images identify irregularities in the magnetic profiles from individual bead populations. Magnetic simulations account for the varying magnetic profiles and allow to infer the magnetization of individual beads. Additionally, this work shows that the imaging technique can be adapted to achieve illumination‐free tracking of magnetic beads, opening the possibility of tracking cell movements and mechanics in photosensitive contexts.     

Can silver nanoparticles be useful as potential biological labels?

Silver (Ag) nanoparticles have unique plasmon-resonant optical scattering properties that are finding use in nanomedical applications such as signal enhancers, optical sensors, and biomarkers. In this study, we examined the chemical and biological properties of Ag nanoparticles of similar sizes, but that differed primarily in their surface chemistry (hydrocarbon versus polysaccharide), in neuroblastoma cells for their potential use as biological labels. We observed strong optical labeling of the cells in a high illumination light microscopy system after 24?h of incubation due to the excitation of plasmon resonance by both types of Ag nanoparticle. Surface binding of both types of Ag nanoparticle to the plasma membrane of the cells was verified with scanning electron microscopy as well as the internalization and localization of the Ag nanoparticles into intracellular vacuoles in thin cell sections with transmission electron microscopy. However, the induction of reactive oxygen species (ROS), degradation of mitochondrial membrane integrity, disruption of the actin cytoskeleton, and reduction in proliferation after stimulation with nerve growth factor were found after incubation with Ag nanoparticles at concentrations of 25?μg?ml(-1) or greater, with a more pronounced effect produced by the hydrocarbon-based Ag nanoparticles in most cases. Therefore, the use of Ag nanoparticles as potential biological labels, even if the surface is chemically modified with a biocompatible material, should be approached with caution.     

Background-free three-dimensional selective imaging of anisotropic plasmonic nanoparticles

There is an increasing demand for advanced optical imaging techniques that can detect and resolve nanosize objects at a spatial resolution below the optical diffraction limit,especially in three-dimensional (3D) cellular environments.In this study,using a polarization-activated localization scheme based on the orientation-dependent properties of anisotropic plasmonic metal nanoparticles (MNPs),"photoswitchable" imaging of single gold nanorods (AuNRs) was accomplished not only in two dimensions but also in three dimensions.Moreover,the Rayleigh scattering background arising from the congested subcellular structures was efficiently suppressed.Thus,we obtained the 3D distributions of both the position and the orientation of the AuNRs inside the cells and investigated their internalization kinetics.To our knowledge,this is the first demonstration of the confocal-like 3D imaging of non-fluorescence nanoparticles with a high resolution and almost zero background.This technique is easy to implement and should greatly facilitate MNP studies and applications in biomedicine and biology.     

New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third‐Harmonic Generation Bioimaging

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth‐inspired synthesis is developed combined with primary mineralization and a ligand‐assisted secondary growth strategy to prepare mesostructured α‐FeOOH nanorods (NRs). The sub‐wavelength heterogeneity of the refractive index leads to enhanced third‐harmonic generation (THG) signals under near‐infrared excited wavelengths at 1230 nm. The as‐prepared NRs exhibit an 11‐fold stronger THG intensity compared to bare α‐FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α‐FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long‐term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe‐based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.     

High Surface‐Enhanced Raman Scattering Performance of Individual Gold Nanoflowers and Their Application in Live Cell Imaging

Molecular imaging techniques based on surface‐enhanced Raman scattering (SERS) face a lack of reproducibility and reliability, thus hampering its practical application. Flower‐like gold nanoparticles have strong SERS enhancement performance due to having plenty of hot‐spots on their surfaces, and this enhancement is not dependent on the aggregation of the particles. These features make this kind of particle an ideal SERS substrate to improve the reproducibility in SERS imaging. Here, the SERS properties of individual flower‐like gold nanoparticles are systematically investigated. The measurements reveal that the enhancement of a single gold nanoparticle is independent of the polarization of the excitation laser with an enhancement factor as high as 10⁸. After capping with Raman signal molecules and folic acid, the gold nanoflowers show strong Raman signal in the living cells, excellent targeting properties, and a high signal‐to‐noise ratio for SERS imaging.     

The Role of Ligand Density and Size in Mediating Quantum Dot Nuclear Transport

Studying the effects of the physicochemical properties of nanomaterials on cellular uptake, toxicity, and exocytosis can provide the foundation for designing safer and more effective nanoparticles for clinical applications. However, an understanding of the effects of these properties on subcellular transport, accumulation, and distribution remains limited. The present study investigates the effects of surface density and particle size of semiconductor quantum dots on cellular uptake as well as nuclear transport kinetics, retention, and accumulation. The current work illustrates that cellular uptake and nuclear accumulation of nanoparticles depend on surface density of the nuclear localization signal (NLS) peptides with nuclear transport reaching a plateau at 20% surface NLS density in as little as 30 min. These intracellular nanoparticles have no effects on cell viability up to 72 h post treatment. These findings will set a foundation for engineering more sophisticated nanoparticle systems for imaging and manipulating genetic targets in the nucleus.     

Enzyme‐Responsive Nanoparticle Systems

Inorganic nanoparticles and their accompanying diverse physical properties are now virtually in routine use as imaging tools in cell‐biology. In addition to serving as excellent contrast agents, their size‐ and environment‐dependent optical and magnetic properties can be harnessed to create enzyme biosensor devices of extremely high sensitivity, whilst circumventing the numerous technical limitations associated with traditional enzyme assays. In this Research News article we discuss recent advances in field of enzyme‐responsive nanoparticle systems, where the activity of an enzyme elicits a specific response in the nanoparticle assembly to produce a signal relating to enzyme activity, focusing on three important systems: DNA‐structured nanoparticles, protein kinases and proteases.     

Wide-Field and Real-Time Super-Resolution Optical Imaging By Titanium Dioxide Nanoparticle-Assembled Solid Immersion Lens

Super-resolution optical imaging techniques can break the optical diffraction limit, thus providing unique opportunities to visualize the microscopic world at the nanoscale. Although near-field optical microscopy techniques have been proven to achieve significantly improved imaging resolution, most near-field approaches still suffer from a narrow field of view (FOV) or difficulty in obtaining wide-field images in real time, which may limit their widespread and diverse applications. Here, the authors experimentally demonstrate an optical microscope magnification and image enhancement approach by using a submillimeter-sized solid immersion lens (SIL) assembled by densely-packed 15 nm TiO₂ nanoparticles through a silicone oil two-step dehydration method. This TiO₂ nanoparticle-assembled SIL can achieve both high transparency and high refractive index, as well as sufficient mechanical strength and easy-to-handle size, thus providing a fast, wide-field, real-time, non-destructive, and low-cost solution for improving the quality of optical microscopic observation of a variety of samples, including nanomaterials, cancer cells, and living cells or bacteria under conventional optical microscopes. This study provides an attractive alternative to simplify the fabrication and applications of high-performance SILs.     

Size‐Dependent Cellular Uptake of DNA Functionalized Gold Nanoparticles

The extensive use of gold nanoparticles (AuNPs) in nanomedicine, especially for intracellular imaging, photothermal therapy, and drug delivery, has necessitated the study of how functionalized AuNPs engage with living biological interfaces like the mammalian cell. Nanoparticle size, shape, surface charge, and surface functionality can affect the accumulation of functionalized AuNPs in cells. Confocal microscopy, flow cytometry, and inductively coupled plasma mass spectrometry demonstrate that CaSki cells, a human cervical cancer cell line, internalize AuNPs functionalized with hairpin, single stranded, and double stranded DNA differently. Surface charge and DNA conformation are shown to have no effect on the cell‐nanoparticle interaction. CaSki cells accumulate small DNA‐AuNPs in greater quantities than large DNA‐AuNPs, demonstrating that size is the major contributor to cellular uptake properties. These data suggest that DNA‐AuNPs can be easily tailored through modulation of size to design functional AuNPs with optimal cellular uptake properties and enhanced performance in nanomedicine applications.     

The translocation of fullerenic nanoparticles into lysosome via the pathway of clathrin-mediated endocytosis

Manufactured fullerene nanoparticles easily enter into cells and hence have been rapidly developed for biomedical uses. However, it is generally unknown which route the nanoparticles undergo when crossing cell membranes and where they localize to the intracellular compartments. Herein we have used both microscopic imaging and biological techniques to explore the processes of [C(60)(C(COOH)(2))(2)](n) nanoparticles across cellular membranes and their intracellular translocation in 3T3 L1 and RH-35 living cells. The fullerene nanoparticles are quickly internalized by the cells and then routed to the cytoplasm with punctate localization. Upon entering the cell, they are synchronized to lysosome-like vesicles. The [C(60)(C(COOH)(2))(2)](n) nanoparticles entering cells are mainly via endocytosis with time-, temperature-?and energy-dependent manners. The cellular uptake of [C(60)(C(COOH)(2))(2)](n) nanoparticles was found to be clathrin-mediated but not caveolae-mediated endocytosis. The endocytosis mechanism and the subcellular target location provide key information for the better understanding and predicting of the biomedical function of fullerene nanoparticles inside?cells.