Pratixa P. Joshi

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.

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.

Magneto-photo-acoustic imaging

Magneto-photo-acoustic imaging, a technique based on the synergy of magneto-motive ultrasound, photoacoustic and ultrasound imaging, is introduced. Hybrid nanoconstructs, liposomes encapsulating gold nanorods and iron oxide nanoparticles, were used as a dual-contrast agent for magneto-photo-acoustic imaging. Tissue-mimicking phantom and macrophage cells embedded in ex vivo porcine tissue were used to demonstrate that magneto-photo-acoustic imaging is capable of visualizing the location of cells or tissues labeled with dual-contrast nanoparticles with sufficient contrast, excellent contrast resolution and high spatial resolution in the context of the anatomical structure of the surrounding tissues. Therefore, magneto-photo-acoustic imaging is capable of identifying the nanoparticle-labeled pathological regions from the normal tissue, providing a promising platform to noninvasively diagnose and characterize pathologies.

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.

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.

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.

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.

Ultrasound-based imaging of nanoparticles: From molecular and cellular imaging to therapy guidance

The effectiveness of an imaging technique is often based on the ability to image quantitatively both morphological and physiological functions of the tissue. Here we present several ultrasound-based imaging techniques capable of visualizing both structural and functional properties of living tissue. Each imaging system utilizes custom-made, targeted nanoparticles developed to probe specific molecular events. Therefore, images of these nanoparticles display molecular processes in the body. Furthermore, the developed nanoparticle contrast agents can also be used for image-guided molecular therapy. For each imaging system, the basic physics and principles behind each approach are described. Experimental aspects of each imaging system including fabrication of integrated imaging probes and associated imaging hardware, and design of targeted contrast agents are discussed. Finally, biomedical and clinical applications of the developed imaging approaches ranging from microscopic to macroscopic imaging of cardiovascular diseases, cancer detection, diagnosis, therapy and therapy monitoring are demonstrated and discussed.

Intravascular photoacoustic imaging of macrophages using molecularly targeted gold nanoparticles

Using contrast agents with desired targeting moiety and optical absorption, intravascular photoacoustic imaging may be used to identify various biomarkers expressed during the progression of atherosclerotic lesions. In this paper, we present intravascular photoacoustic imaging of macrophages in the atherosclerotic lesions using bio-conjugated gold nanoparticles as the contrast agent. Atherosclerotic lesions were created in the aorta of a New Zealand white rabbit subjected to a high cholesterol diet and balloon injury. The rabbit was injected with 20 nm spherical gold nanoparticles conjugated with antibodies. The macrophages with internalized gold nanoparticles were imaged by intravascular photoacoustic imaging in the near infrared range; this was possible because of plasmon resonance coupling between closely spaced gold nanoparticles internalized by macrophages. The multi-wavelength intravascular photoacoustic images of the diseased aorta were analyzed to identify the presence and location of macrophages labeled with gold nanoparticles. Spectroscopic intravascular photoacoustic image showing the distribution of gold nanoparticles was further confirmed by the gold-specific silver staining of the tissue crosssection. The results of our study suggest that molecular intravascular photoacoustic imaging can be used to image macrophages in atherosclerosis.

On stability of molecular therapeutic agents for noninvasive photoacoustic and ultrasound image-guided photothermal therapy

Image-guided molecular photothermal therapy using targeted gold nanoparticles acting as photoabsorbers can be used to noninvasively treat various medical conditions including cancer. Among different types of gold nanoparticles, gold nanorods are an attractive candidate for both photothermal therapy and photoacoustic imaging due to their high and tunable optical absorption cross-section. However, nanorods are not thermodynamically stable; under laser exposure, the nanorods can easily transform to spheres, thus changing their desired optical properties. In this study, gold-silica coreshell nanorods were prepared by coating silica directly onto the surface of PEGylated gold nanorods using a modified Stöber method. The nanorods were exposed to 800 nm wavelength, 7 ns pulses of light at a 10 Hz pulse repetition rate. For different fluences ranging from 0 to 8 mJ/cm2, the optical extinction spectrum was measured before and after the exposure to investigate their photothermal stability. Finally, the effectiveness of gold-silica core-shell nanoparticles as a photoacoustic contrast agent and photothermal nanoabsorber was tested using inclusion-embedded phantoms and a combined ultrasound and photoacoustic imaging system. The results of our study suggest that gold-silica core-shell nanorods are excellent candidates for image-guided molecular photothermal therapy.