Visualization of molecular composition and functionality of cancer cells using nanoparticle-augmented ultrasound-guided photoacoustics.

Assessment of molecular signatures of tumors in addition to their anatomy and morphology is desired for effective diagnostic and therapeutic procedures. Development of in vivo imaging techniques that can identify and monitor molecular composition of tumors remains an important challenge in pre-clinical research and medical practice. Here we present a molecular photoacoustic imaging technique that can visualize the presence and activity of an important cancer biomarker - epidermal growth factor receptor (EGFR), utilizing the effect of plasmon resonance coupling between molecular targeted gold nanoparticles. Specifically, spectral analysis of photoacoustic images revealed profound changes in the optical absorption of systemically delivered EGFR-targeted gold nanospheres due to their molecular interactions with tumor cells overexpressing EGFR. In contrast, no changes in optical properties and, therefore, photoacoustic signal, were observed after systemic delivery of non-targeted gold nanoparticles to the tumors. The results indicate that multi-wavelength photoacoustic imaging augmented with molecularly targeted gold nanoparticles has the ability to monitor molecular specific interactions between nanoparticles and cell-surface receptors, allowing visualization of the presence and functional activity of tumor cells. Furthermore, the approach can be used for other cancer cell-surface receptors such as human epidermal growth factor receptor 2 (HER2). Therefore, ultrasound-guided molecular photoacoustic imaging can potentially aid in tumor diagnosis, selection of customized patient-specific treatment, and monitor the therapeutic progression and outcome in vivo.

Use of colloidal quantum dots as a digitally switched swept light source for gold nanoparticle based hyperspectral microscopy.

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.

Development and optimization of near-IR contrast agents for immune cell tracking.

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.

Conjugation of antibodies to gold nanorods through Fc portion: synthesis and molecular specific imaging.

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.

Photoacoustic imaging: a potential tool to detect early indicators of metastasis.

The metastasis of cancer is a multistage process involving complex biological interactions and difficult to predict outcomes. Accurate assessment of the extent of metastasis is critical for clinical practice; unfortunately, medical imaging methods capable of identifying the early stages of invasion and metastasis are lacking. Photoacoustic imaging is capable of providing noninvasive, real-time imaging of significant anatomical and physiological changes. indicating the progression of cancer invasion and metastasis. Preclinically, photoacoustic methods have been used to image lymphatic anatomy, including the sentinel lymph nodes, to identify circulating tumor cells within vasculature and to detect micrometastases. Progress has begun toward the development of clinically applicable photoacoustic imaging systems to assist with the determination of cancer stage and likelihood of metastatic invasion.

Preventing protein adsorption and macrophage uptake of gold nanoparticles via a hydrophobic shield.

Polyethylene glycol (PEG) surface coatings are widely used to render stealth properties to nanoparticles in biological applications. There is abundant literature on the benefits of PEG coatings and their ability to reduce protein adsorption, to diminish nonspecific interactions with cells, and to improve pharmacokinetics, but very little discussion of the limitations of PEG coatings. Here, we show that physiological concentrations of cysteine and cystine can displace methoxy-PEG-thiol molecules from the gold nanoparticle (GNP) surface that leads to protein adsorption and cell uptake in macrophages within 24 h. Furthermore, we address this problem by incorporating an alkyl linker between the PEG and the thiol moieties that provides a hydrophobic shield layer between the gold surface and the hydrophilic outer PEG layer. The mPEG-alkyl-thiol coating greatly reduces protein adsorption on GNPs and their macrophage uptake. This has important implications for the design of GNP for biological systems.

On sensitivity of molecular specific photoacoustic imaging using plasmonic gold nanoparticles.

Functionalized gold nanospheres undergo receptor mediated aggregation on cancer cells that overexpress the epidermal growth factor receptor (EGFR). This phenomenon leads to a red shift in the plasmon resonance frequency of the EGFR-targeted gold nanoparticles. Previously we demonstrated that highly selective detection of cancer cells can be achieved using the combination of multi-wavelength photoacoustic imaging and molecular specific gold nanoparticles. In this study, we use tissue models to evaluate the sensitivity of molecular specific photoacoustic imaging in detecting cancer cells labeled with EGFR-targeted gold nanoparticles. The results of our study indicate that highly sensitive detection of cancer cells is feasible using photoacoustic imaging and plasmonic gold nanoparticles.

Molecular therapeutic agents for noninvasive photoacoustic image-guided photothermal therapy.

Gold nanoparticles are attracting increasing attention in nanomedicine due to their inherently low toxicity and unique optical properties. In particular, gold nanorods have been used in the thermal therapy due to their tunable strong longitudinal plasmon resonance in the near infra-red region and high conversion efficiency from optical to thermal energy. In this study we explore the potential of gold nanorods for photoacoustic image-guided photothermal therapy to treat cancers. We synthesize the gold nanorods and make them biocompatible by replacing the cytotoxic surfactant used in the synthesis (cetyl trimethyl ammonium bromide) with a biocompatible molecule and then demonstrate the targeting to the cancer cells by bioconjugation of the modified nanorods.

Multiwavelength photoacoustic imaging and plasmon resonance coupling of gold nanoparticles for selective detection of cancer.

Gold nanoparticles targeting epidermal growth factor receptor via antibody conjugation undergo molecular specific aggregation when they bind to receptors on cell surfaces, leading to a red shift in their plasmon resonance frequency. Capitalizing on this effect, we demonstrate the efficacy of the molecular specific photoacoustic imaging technique using subcutaneous tumor-mimicking gelatin implants in ex-vivo mouse tissue. The results of our study suggest that highly selective and sensitive detection of cancer cells is possible using multiwavelength photoacoustic imaging and molecular specific gold nanoparticles.

Buckysomes: fullerene-based nanocarriers for hydrophobic molecule delivery.

We report the preparation and preliminary in vitro studies of nanocarriers termed "buckysomes," which are self-assembled, spherical nanostructures composed of the amphiphilic fullerene AF-1. By inducing AF-1 self-assembly at an elevated temperature of 70 degrees C, dense spherical buckysomes with diameters of 100-200 nm were formed, as observed by electron microscopy and dynamic light scattering. The amphiphilic nature of AF-1 results in the formation of many hydrophobic regions within the buckysomes, making them ideal for embedding hydrophobic molecules to be tested in a drug delivery scheme. After confirming the cellular internalization of buckysomes embedded with the hydrophobic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, we embedded paclitaxel, a highly hydrophobic anticancer drug. The in vitro therapeutic efficacy of the paclitaxel-embedded buckysomes toward suppression of MCF-7 breast cancer cell growth was compared to that of Abraxane, a commercially available, nanoparticle-albumin-bound formulation of paclitaxel. Notably, the paclitaxel-embedded buckysomes demonstrated a similar efficacy to that observed with Abraxane in cell viability studies; these results were confirmed microscopically. Moreover, negative control studies of MCF-7 viability using empty buckysomes demonstrated that the buckysomes were not cytotoxic. The results of our studies suggest that buckysomes prepared from self-assembly of AF-1 at 70 degrees C are promising nanomaterials for the delivery of hydrophobic molecules.

Nanostructured biosensors built by layer-by-layer electrostatic assembly of enzyme-coated single-walled carbon nanotubes and redox polymers.

In this study, we describe the construction of glucose biosensors based on an electrostatic layer-by-layer (LBL) technique. Gold electrodes were initially functionalized with negatively charged 11-mercaptoundecanoic acid followed by alternate immersion in solutions of a positively charged redox polymer, poly[(vinylpyridine)Os(bipyridyl)2Cl(2+/3+)], and a negatively charged enzyme, glucose oxidase (GOX), or a GOX solution containing single-walled carbon nanotubes (SWNTs). The LBL assembly of the multilayer films were characterized by UV-vis spectroscopy, ellipsometry, and cyclic voltammetry, while characterization of the single-walled nanotubes was performed with transmission electron microscopy, Raman spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy. When the GOX solution contained single-walled carbon nanotubes (GOX-SWNTs), the oxidation peak currents during cyclic voltammetry increased 1.4-4.0 times, as compared to films without SWNTs. Similarly the glucose electro-oxidation current also increased (6-17 times) when SWNTs were present. By varying the number of multilayers, the sensitivity of the sensors could be controlled.

Amperometric biosensors based on redox polymer-carbon nanotube-enzyme composites.

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl(2+/3+)] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to approximately 1 mA/cm(2) for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.