Jesse Aaron

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties

Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is ∼6 h.

Optical Systems for in Vivo Molecular Imaging of Cancer

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Molecular specific optoacoustic imaging with plasmonic nanoparticles

Gold nanoparticles functionalized with antibodies can specifically bind to molecular biomarkers such as epithelial growth factor receptor (EGFR). The molecule specific nature of the antibody-functionalized gold nanoparticles forms the basis for the developed optoacoustic imaging technique to detect cancer at an asymptotic stage. Optoacoustic imaging was performed with 532 nm and 680 nm pulsed laser irradiation on three-dimensional tissue phantoms prepared using a human keratinocyte cell line. The results of our study demonstrate that the combination of anti-EGFR gold ioconjugates and optoacoustic imaging can allow highly sensitive and selective detection of human epithelial cancer cells.

Plasmon resonance coupling of metal nanoparticles for molecular imaging of carcinogenesis in vivo

An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.

Dynamic Imaging of Molecular Assemblies in Live Cells Based on Nanoparticle Plasmon Resonance Coupling

We used molecular-specific gold nanoparticles to monitor epidermal growth factor receptors (EGFR) in live A431 cells over time. Dark-field hyperspectral imaging, electron microscopy, and electrodynamic modeling were used to correlate optical properties of EGFR-bound plasmonic nanoparticles with receptor regulation state. We showed that receptor trafficking resulted in a progressive red shift of greater than 100 nm in the nanoparticle plasmon resonance wavelength over a time period of 60 min. Furthermore, we demonstrated that changes in peak scattering wavelengths of gold nanoparticles from 546 +/- 15 to 574 +/- 20, and to 597 +/- 44 nm are associated with EGFR trafficking from the cell membrane, to early endosomes, and to late endosomes/multivesicular bodies, respectively. Finally, we used the changes in scattering spectra of EGFR-bound nanoparticles and a straightforward statistical analysis of RGB-channel color images of labeled cells to create near real-time maps of EGFR regulatory states in living cells.

Hybrid plasmonic magnetic nanoparticles as molecular specific agents for MRI/optical imaging and photothermal therapy of cancer cells

Nanoparticles which consist of a plasmonic layer and an iron oxide moiety could provide a promising platform for development of multimodal imaging and therapy approaches in future medicine. However, the feasibility of this platform has yet to be fully explored. In this study we demonstrated the use of gold-coated iron oxide hybrid nanoparticles for combined molecular specific MRI/optical imaging and photothermal therapy of cancer cells. The gold layer exhibits a surface plasmon resonance that provides optical contrast due to light scattering in the visible region and also presents a convenient surface for conjugating targeting moieties, while the iron oxide cores give strong T2 (spin?spin relaxation time) contrast. The strong optical absorption of the plasmonic gold layer also makes these nanoparticles a promising agent for photothermal therapy. We synthesized hybrid nanoparticles which specifically target epidermal growth factor receptor (EGFR), a common biomarker for many epithelial cancers. We demonstrated molecular specific MRI and optical imaging in MDA-MB-468 breast cancer cells. Furthermore, we showed that receptor-mediated aggregation of anti-EGFR hybrid nanoparticles allows selective destruction of highly proliferative cancer cells using a nanosecond pulsed laser at 700?nm wavelength, a significant shift from the peak absorbance of isolated hybrid nanoparticles at 532?nm.

Polarization microscopy with stellated gold nanoparticles for robust, in-situ monitoring of biomolecules

Advances in plasmonic nanoparticle synthesis afford new opportunities for biosensing applications. Here, we apply a combination of a new type of plasmonic nanomaterial – stellated nanoparticles, and polarization-sensitive darkfield microscopy for detecting molecular assemblies and tracking of individual epidermal growth factor receptors within single live cells with high signal-to-background ratio. Depolarization of linear polarized light by stellated nanoparticles is over 15-fold more efficient than similarly-sized spheroidal nanoparticles. This efficient light depolarization allows robust detection of molecules labeled with stellated nanoparticles in cross-polarized imaging where the intrinsic light scattering from cells is significantly reduced. The imaging can be carried out with single molecule sensitivity for essentially unlimited time with no signal degradation.

Optical sectioning using a fiber probe with an angled illumination-collection geometry: evaluation in engineered tissue phantoms

We present a fiber optic probe that combines polarized illumination and detection with an angled distal probe geometry to detect the size-dependent scattering at a specific depth within epithelium. Analysis of the scattering signal by use of Mie theory allows the extraction of scatterer size and size distribution-key parameters for precancer detection. The probe was evaluated in two tissue phantoms: polystyrene beads atop collagen gel and multiple layers of cancer cells atop collagen. We also present in vivo measurements in the oral cavity of normal volunteers. The sizes of scatterers extracted from the scattering spectra corresponded to independently measured values.

EGFR-Targeted Hybrid Plasmonic Magnetic Nanoparticles Synergistically Induce Autophagy and Apoptosis in Non-Small Cell Lung Cancer Cells

Background The epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells. Methodology/Principal Findings Cell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity. Conclusions/Significance We demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in lung cancer cells that are not characteristic for free molecular therapeutics thus increasing efficacy of therapy against NSCLC.

Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles

We describe a new approach for optical imaging that combines the advantages of molecularly targeted plasmonic nanoparticles and magnetic actuation. This combination is achieved through hybrid nanoparticles with an iron oxide core surrounded by a gold layer. The nanoparticles are targeted in-vitro to epidermal growth factor receptor, a common cancer biomarker. The gold portion resonantly scatters visible light giving a strong optical signal and the superparamagnetic core provides a means to externally modulate the optical signal. The combination of bright plasmon resonance scattering and magnetic actuation produces a dramatic increase in contrast in optical imaging of cells labeled with hybrid gold/iron oxide nanoparticles.