Sensitive and quantitative assessment of changes in circulating tumor cells (CTCs) can help in cancer prognosis and in the evaluation of therapeutics efficacy. However, extremely low occurrence of CTCs in the peripheral blood (approximately one CTC per billion blood cells) and potential changes in molecular biomarkers during the process of epithelial to mesenchymal transition (EMT) create technical hurdles to the enrichment and enumeration CTCs. Recently, efforts have been directed toward development of antibody-capture assays based on the expression of the common biomarker - the epithelial cell adhesion molecule (EpCAM) of epithelium-derived cancer cells. Despite some promising results, the assays relying on EpCAM capture have shown inconsistent sensitivity in clinical settings and often fail to detect CTCs in patients with metastatic cancer. We have addressed this problem by the development of an assay based on hybrid magnetic/plasmonic nanocarriers and a microfluidic channel. In this assay cancer cells are specifically targeted by antibody-conjugated magnetic nanocarriers and are separated from normal blood cells by a magnetic force in a microfluidic chamber. Subsequently, immunofluorescence staining is used to differentiate CTCs from normal blood cells. We demonstrated in cell models of colon, breast and skin cancers that this platform can be easily adapted to a variety of biomarkers, targeting both surface receptor molecules and intracellular biomarkers of epithelial-derived cancer cells. Experiments in whole blood showed capture efficiency greater than 90% when two cancer biomarkers are used for cell capture. Thus, the combination of immunotargeted magnetic nanocarriers with microfluidics provides an important platform that can improve the effectiveness of current CTC assays by overcoming the problem of heterogeneity of tumor cells in the circulation.
gold shell/magnetic core nanoparticles; circulating tumor cells; immunomagnetic assay; microfluidic chip
Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross-sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection.
Gold nanorods; molecular conjugation; molecular specific imaging; targeted nanoparticles
The adsorption of even a single serum protein molecule on a gold nanosphere used in biomedical imaging may increase the size too much for renal clearance. Herein, we design charged ~5 nm Au nanospheres coated with binary mixed charge ligand monolayers that do not change in size upon incubation in pure fetal bovine serum (FBS). This lack of protein adsorption is unexpected given the Au surface is moderately charged. The mixed charge monolayers are comprised of anionic citrate ligands modified by place exchange with naturally-occurring amino acids: either cationic lysine or zwitterionic cysteine ligands. The zwitterionic tips of either the lysine or cysteine ligands interact weakly with the proteins and furthermore increase the distance between the “buried” charges closer to the Au surface and the interacting sites on the protein surface. The ~5 nm nanospheres were assembled into ~20 nm diameter nanoclusters with strong NIR absorbance (of interest in biomedical imaging and therapy) with a biodegradable polymer, PLA(1k)-b-PEG(10k)-b-PLA(1k). Upon biodegradation of the polymer in acidic solution, the nanoclusters dissociated into primary ~5 nm Au nanospheres, which also did not adsorb any detectable serum protein in undiluted FBS.
We propose a method to utilize colloidal quantum dots (QDs) as a swept light source for hyperspectral microscopy. The use of QD allows for uniform multicolor emission which covers visible-NIR wavelengths. We used 8 colors of CdSe/ZnS and CdTe/ZnS colloidal quantum dots with the peak emission wavelengths from 520 nm to 800 nm. The QDs are packed in a compact enclosure, composing a low-cost, solid-state swept light source that can be easily used in most microscopes. Multicolor emission from the QDs is simply controlled by digitally switching excitation UVLEDs, eliminating the use of mechanically-driven gratings or filters. We used gold nanoparticles as optical markers for hyperspectral microscopy. Due to the effect of localized surface plasmon resonance, gold nanoparticles demonstrate size and shape-dependent absorption spectra. Employed in a standard microscope, the QD light source enabled multispectral absorption imaging of macrophage cells labeled with gold nanorods and nanospheres.
(170.0110) Imaging systems; (110.4234) Multispectral and hyperspectral imaging; (300.6550) Spectroscopy, visible; (170.1530) Cell analysis; (160.4236) Nanomaterials; (230.3670) Light-emitting diodes
Although sub-100 nm nanoclusters of metal nanoparticles are of interest in many fields including biomedical imaging, sensors and catalysis, it has been challenging to control their morphologies and chemical properties. Herein, a new concept is presented to assemble equilibrium Au nanoclusters of controlled size by tuning the colloidal interactions with a polymeric stabilizer, PLA(1k)-b-PEG(10k)-b-PLA(1k). The nanoclusters form upon mixing a dispersion of ~5 nm Au nanospheres with a polymer solution followed by partial solvent evaporation. A weakly adsorbed polymer quenches the equilibrium nanocluster size and provides steric stabilization. Nanocluster size is tuned from ~20 nm to ~40 nm by experimentally varying the final Au nanoparticle concentration and the polymer/Au ratio, along with the charge on the initial Au nanoparticle surface. Upon biodegradation of the quencher, the nanoclusters reversibly and fully dissociate to individual ~5 nm primary particles. Equilibrium cluster size is predicted semi-quantitatively with a free energy model that balances short-ranged depletion and van der Waals attractions with longer-ranged electrostatic repulsion, as a function of the Au and polymer concentrations. The close spacings of the Au nanoparticles in the clusters produce strong NIR extinction over a broad range of wavelengths from 650 to 900 nm, which is of practical interest in biomedical imaging.
nanoclusters; plasmonic nanoparticles; colloidal forces; depletion attraction; biodegradable nanoparticles; equilibrium assembly
The photothermal stability of plasmonic nanoparticles is critically important to perform reliable photoacoustic imaging and photothermal therapy. Recently, biodegradable nanoclusters composed of sub-5 nm primary gold particles and a biodegradable polymer have been reported as clinically-translatable contrast agents for photoacoustic imaging. After cellular internalization, the nanoclusters degrade into 5 nm primary particles for efficient excretion from the body. In this paper, three different sizes of biodegradable nanoclusters were synthesized and the optical properties and photothermal stability of the nanoclusters were investigated and compared to that of gold nanorods. The results of our study indicate that 40 nm and 80 nm biodegradable nanoclusters demonstrate higher photothermal stability compared to gold nanorods. Furthermore, 40 nm nanoclusters produce higher photoacoustic signal than gold nanorods at a given concentration of gold. Therefore, the biodegradable plasmonic nanoclusters can be effectively used for photoacoustic imaging and photothermal therapy.
(170.5120) Photoacoustic imaging; (170.3880) Medical and biological imaging
A newly selected anti-receptor (anti-EGFR) aptamer was conjugated to gold nanoparticles via a facile hybridization method and was found to specifically and quantitatively direct the delivery of gold nanoparticles to cells expressing EGFR through receptor-mediated endocytosis.
Clusters of metal nanoparticles with an overall size less than 100 nm and high metal loadings for strong optical functionality, are of interest in various fields including microelectronics, sensors, optoelectronics and biomedical imaging and therapeutics. Herein we assemble ~5 nm gold particles into clusters with controlled size, as small as 30 nm and up to 100 nm, which contain only small amounts of polymeric stabilizers. The assembly is kinetically controlled with weakly adsorbing polymers, PLA(2K)-b-PEG(10K)-b-PLA(2K) or PEG (MW = 3350), by manipulating electrostatic, van der Waals (VDW), steric, and depletion forces. The cluster size and optical properties are tuned as a function of particle volume fractions and polymer/gold ratios to modulate the interparticle interactions. The close spacing between the constituent gold nanoparticles and high gold loadings (80–85% w/w gold) produce a strong absorbance cross section of ~9×10−15 m2 in the NIR at 700 nm. This morphology results from VDW and depletion attractive interactions that exclude the weakly adsorbed polymeric stabilizer from the cluster interior. The generality of this kinetic assembly platform is demonstrated for gold nanoparticles with a range of surface charges from highly negative to neutral, with the two different polymers.
Gold nanorods (NRs) are attractive for in vivo imaging due to their high optical cross-sections and tunable absorbance. However, the feasibility of using NRs for cell tracking has not been fully explored. Here, we synthesized dye doped silica-coated NRs as multimodal contrast agents for imaging of macrophages – immune cells which play an important role in cancer and cardiovascular diseases. We showed the importance of silica coating in imaging of NR-labeled cells. Photoacoustic (PA) imaging of NRs labeled macrophages showed high sensitivity. Therefore, these results provide foundation for applications of silica-coated NRs and PA imaging in tracking of immune cells.
(170.0170) Medical optics and biotechnology; (170.5120) Photoacoustic imaging; (170.2655) Functional monitoring and imaging; (170.2520) Fluorescence microscopy; (060.4230) Multiplexing; (160.4236) Nanomaterials
As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular trafficking of nanoparticles – an important part of cell-nanoparticle interaction, has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique – pulsed magneto-motive ultrasound (pMMUS), to identify intracellular trafficking of endocytosed magnetic nanoparticles. In pulsed magneto-motive ultrasound imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular aggregation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular trafficking non-invasively and in real-time.
Pulsed magneto-motive ultrasound imaging; superparamagnetic iron-oxide nanoparticles; macrophage; endocytosis; intracellular trafficking
Metal nanoparticles with surface plasmon resonance (SPR) in the near-infrared region (NIR) are of great interest for imaging and therapy. Presently, gold nanoparticles with NIR absorbance are typically larger than 50nm, above the threshold size of ~5 nm required for efficient renal clearance. As these nanoparticles are not biodegradable, concerns about long-term toxicity have restricted their translation into the clinic. Here, we address this problem by developing a flexible platform for the kinetically-controlled assembly of sub-5 nm ligand-coated gold particles to produce metal/polymer biodegradable nanoclusters smaller than 100 nm with strong NIR absorbance for multimodal application. A key novel feature of the proposed synthesis is the use of weakly adsorbing biodegradable polymers that allows tight control of nanocluster size and, in addition, results in nanoclusters with unprecedented metal loadings, and thus optical functionality. Over time, the biodegradable polymer stabilizer degrades under physiological conditions that leads to disassembly of the nanoclusters into sub-5nm primary gold particles which are favorable for efficient body clearance. This synthesis of polymer/inorganic nanoclusters combines the imaging contrast and therapeutic capabilities afforded by the NIR-active nanoparticle assembly with the biodegradability of a polymer stabilizer.
plasmonic nanoparticles; biodegradation of nanoparticles; near-infrared nanoparticles; clearance of nanoparticles; nanoclusters
Plasmonic metal nanoparticles are used in photoacoustic imaging as contrast agents because of their resonant optical absorption properties in the visible and near-IR regions. However, the nanoparticles could accumulate and result in long-term toxicity in vivo, because they are generally not biodegradable. Recently, biodegradable plasmonic gold nanoclusters, consisting of sub-5 nm primary gold nanoparticles and biodegradable polymer stabilizer, were introduced. In this Letter, we demonstrate the feasibility of biodegradable nanoclusters as a photoacoustic contrast agent. We performed photoacoustic and ultrasound imaging of a tissue-mimicking phantom with inclusions containing nanoclusters at various concentrations. The results indicate that the biodegradable gold nanoclusters can be used as effective contrast agents in photoacoustic imaging.
Intravascular photoacoustic (IVPA) imaging is a catheter-based, minimally invasive, imaging modality capable of providing high-resolution optical absorption map of the arterial wall. Integrated with intravascular ultrasound (IVUS) imaging, combined IVPA and IVUS imaging can be used to detect and characterize atherosclerotic plaques building up in the inner lining of an artery. In this paper, we present and discuss various representative applications of combined IVPA/IVUS imaging of atherosclerosis, including assessment of the composition of atherosclerotic plaques, imaging of macrophages within the plaques, and molecular imaging of biomarkers associated with formation and development of plaques. In addition, imaging of coronary artery stents using IVPA and IVUS imaging is demonstrated. Furthermore, the design of an integrated IVUS/IVPA imaging catheter needed for in vivo clinical applications is discussed.
Atherosclerosis; contrast agent; imaging catheter; intravascular photoacoustic (IVPA) imaging; intravascular ultrasound (IVUS) imaging; molecular imaging; stent; vulnerable plaque
Gold (Au) nanoshells were grown on silica nanoparticles with differing average diameters, ranging from 30 nm to 120 nm. Au nanoshells were also formed on silica spheres encapsulating 5 nm diameter magnetic iron oxide nanocrystals. The optical absorbance spectra of these Au nanoshells are reported. The plasmon resonance wavelengths of the smaller diameter nanoshells were significantly less tunable than those of the larger diameter nanoshells. This is due to a reduced range of accessible core-shell ratio—the geometric factor that determines the plasmon peak position—as the silica core diameter shrinks. The smaller diameter nanoshells were also found to be highly prone to aggregation, which broadens the plasmon absorption peak. Model calculations of dispersion stability as a function of silica core diameter reveal that smaller diameter Au shells exhibit more aggregation because of the size-dependence of the electrostatic double-layer potential.
Gold nanorods can be used as extremely bright labels for differential light scattering measurements using two closely spaced wavelengths, thereby detecting human disease through several centimeters of tissue in vivo. They have excellent biocompatibility, are non-toxic, and are not susceptible to photobleaching. They have narrow, easily tunable plasmon spectral lines and thus can image multiple molecular targets simultaneously. Because of their small size, gold nanorods can be transported to various tissues inside the human body via the vasculature and microvasculature, and since they are smaller than vascular pore sizes, they can easily cross vascular space and enter individual cells.
(110.0113) Imaging through turbid media; (170.6510) Spectroscopy, tissue diagnostics
Application of photothermal Optical Coherence Tomography (OCT) to detect macrophages in ex vivo rabbit arteries which have engulfed nanoclusters of gold coated iron oxide (nanorose) is reported. Nanorose engulfed by macrophages associated with atherosclerotic lesions in rabbit arteries absorb incident laser (800nm) energy and cause optical pathlength (OP) variation which is measured using photothermal OCT. OP variation in polydimethyl siloxane tissue phantoms containing varying concentrations of nanorose match values predicted from nanoparticle and material properties. Measurement of OP variation in rabbit arteries in response to laser excitation provides an estimate of nanorose concentration in atherosclerotic lesions of 2.5x109 particles/ml. OP variation in atherosclerotic lesions containing macrophages taking up nanorose has a different magnitude and profile from that observed in control thoracic aorta without macrophages and is consistent with macrophage presence as identified with RAM-11 histology staining. Our results suggest that tissue regions with macrophages taking up nanorose can be detected using photothermal OCT.
(170.4500) Optical Coherence Tomography; (170.3880) Medical and Biological Imaging