Detecting and destroying cancer cells in more than one way with noble metals and different confinement properties on the nanoscale

Abstract

Today, 1 in 2 males and 1 in 3 females in the United States will develop cancer at some point during their lifetimes, and 1 in 4 males and 1 in 5 females in the United States will die from the disease. New methods for detection and treatment have dramatically improved cancer care in the United States. However, as improved detection and increasing exposure to carcinogens has led to higher rates of cancer incidence, clinicians and researchers have not balanced that increase with a similar decrease in cancer mortality rates. This mismatch highlights a clear and urgent need for increasingly potent and selective methods with which to detect and treat cancers at their earliest stages. Nanotechnology, the use of materials with structural features ranging from 1 to 100 nm in size, has dramatically altered the design, use, and delivery of cancer diagnostic and therapeutic agents. The unique and newly discovered properties of these structures can enhance the specificities with which biomedical agents are delivered, complementing their efficacy or diminishing unintended side effects. Gold (and silver) nanotechnologies afford a particularly unique set of physiological and optical properties which can be leveraged in applications ranging from in vitro/vivo therapeutics and drug delivery to imaging and diagnostics, surgical guidance, and treatment monitoring. Nanoscale diagnostic and therapeutic agents have been in use since the development of micellar nanocarriers and polymer-drug nanoconjugates in the mid-1950s, liposomes by Bangham and Watkins in the mid-1960s, and the introduction of polymeric nanoparticles by Langer and Folkman in 1976. Since then, nanoscale constructs such as dendrimers, protein nanoconjugates, and inorganic nanoparticles have been developed for the systemic delivery of agents to specific disease sites. Today, more than 20 FDA-approved diagnostic or therapeutic nanotechnologies are in clinical use with roughly 250 others in clinical development. The global market for nano-enabled medical technologies is expected to grow to $70-160 billion by 2015, rivaling the current market share of biologics worldwide. In this Account, we explore the emerging applications of noble metal nanotechnologies in cancer diagnostics and therapeutics carried out by our group and by others. Many of the novel biomedical properties associated with gold and silver nanoparticles arise from confinement effects: (i) the confinement of photons within the particle which can lead to dramatic electromagnetic scattering and absorption (useful in sensing and heating applications, respectively); (ii) the confinement of molecules around the nanoparticle (useful in drug delivery); and (iii) the cellular/subcellular confinement of particles within malignant cells (such as selective, nuclear-targeted cytotoxic DNA damage by gold nanoparticles). We then describe how these confinement effects relate to specific aspects of diagnosis and treatment such as (i) laser photothermal therapy, optical scattering microscopy, and spectroscopic detection, (ii) drug targeting and delivery, and (iii) the ability of these structures to act as intrinsic therapeutic agents which can selectively perturb/inhibit cellular functions such as division. We intend to provide the reader with a unique physical and chemical perspective on both the design and application of these technologies in cancer diagnostics and therapeutics. We also suggest a framework for approaching future research in the field.

Figure 1. Properties of gold nanotechnologies that can be used to enhance therapeutic treatment and diagnostic imaging of cancer

(a) Photothermal therapy: gold nanoparticles can serve as contrast agents for the selective laser photothermal ablation of tumor cells. Arrow indicates laser focus. Image obtained in collaboration with Prof. X. Huang (U of Memphis) and Prof. C.K. Payne (Georgia Tech). (b) Photoacoustic cytometry/tomography: pulsed laser excitation of cells/tissues labeled with gold nanoparticles can be used to detect or sequester circulating tumor cells (CTCs, upper panel) or to non-invasively image/diagnose/stage tumors and guide surgical procedures (lower panel). (c) Optical coherence tomography (OCT): the backscattering and photothermal properties of gold nanotechnologies can be used to enhance OCT contrast for monitoring disease metastasis to the lymphatic system. (d) Surface enhanced Raman scattering (SERS): Electromagnetic near-field enhancements generated by gold nanoparticles can improve non-invasive in vitro (upper panel) and in vivo (lower panel) spectral cancer diagnostics. Adapted with permission from (b) Refs. ^,, (c) Ref. , and (d) Refs. ^,. Copyright (b) 2009 Macmillan Publishers Ltd: Nature Nanotechnology and 2010 American Chemical Society, (c) 2011 American Chemical Society, and (d) 2007 American Chemical Society and 2009 National Academy of Sciences.

Figure 2. In vitro applications of targeted gold nanotechnologies in diagnostic imaging and photothermal therapy

(a) High optical scattering contrast from malignant cells selectively labeled with antibody-conjugated gold nanospheres and nanorods (anti-epidermal growth factor receptor, anti-EGFR). (b) Specific-labeling by these particles significantly lowers the laser power exposure threshold required to kill malignant, but not normal, cells. Adapted with permission from Ref. . Copyright 2006 American Chemical Society.

Figure 3. The first report demonstrating the use of gold nanorods as contrast agents for laser photothermal cancer therapy

(a) Near-infrared (NIR) transmission image of tumor-bearing mice (rear flank) showing gold-nanorod contrast agents specifically accumulated at the tumor site (dark, highly absorbing region). (b) Thermal transient measurements from the tumor center during NIR laser exposure of gold nanorod-loaded tumors treated by intravenous (left) and intratumoral (right) administration. High thermal contrast is observed between particle-treated mice (red curves) and controls treated by laser only (blue). (c) Tumor regression following intravenous (green) and intratumoral (red) laser photothermal treatment as compared to untreated controls (blue). No detectable disease was observed at day 13 in >50% of the interstitially-treated group and 25% of the intravenously-treated group. (d) Dramatic tumor growth remission/resorption even in a weakly responding mouse treated by intratumoral nanorod injection (electron microscopy, inset) and laser photothermal therapy versus sham (laser alone) treatment. Adapted with permission from Ref. . Copyright 2008 Elsevier Science B.V..

Figure 4. Effects of active targeting on the in vitro uptake and in vivo pharmacokinetics/biodistribution of anticancer gold nanorods

(a) Electron micrographs of near-infrared absorbing colloidal gold nanorods and (b) their corresponding distribution of hydrodynamic diameters. (c) Enhanced in vitro binding/uptake of actively targeted gold nanorods as determined by mass spectrometry (ICP). In vitro accumulation of single-chain variable fragment (ScFv), peptide fragment (ATF), and cyclic cell-penetrating peptide (c-RGD) targeted nanorods was significantly enhanced. In contrast, in vivo (d) pharmacokinetics, (e) blood half-lives, (f) biodistribution, and (g) tumor-specific accumulation was only marginally enhanced. Adapted with permission from Ref. . Copyright 2010 American Chemical Society.

Figure 5. Applications of targeted gold nanotechnologies for spectral cancer diagnostics

(a) Structure of a synthetic nuclear-localization sequence (NLS) peptide derivative which (b) promotes cellular uptake and cancer-selective perinuclear localization of gold nanorod bioconjugates. (c) Optical spectroscopy of immunolabeled gold nanorods (anti-EGFR) showing low surface enhanced Raman scattering (SERS) from normal cells and a high degree of cancer-specific spectral features caused by the high density of SERS-enhancing particles labeling malignant cells. (e) High-density orientation of the nanorods about the malignant cell surface leads to polarized Raman scattering intensity from the vibrations of molecules on the nanorod surfaces. Adapted with permission from (a-b) Ref. and (c-e) Ref. . Copyright 2007 American Chemical Society.

Figure 6. Properties of nanoscale technologies that can be leveraged to enhance the diagnosis and treatment of solid tumors

(a) The EPR effect: Polymer cast replicas of blood vessels in normal (left) and malignant tissues (right) illustrate why high molecular weight compounds (i.e. nanoparticles) can preferentially accumulate at tumor sites supplied by disordered vasculatures. (b) Multivalent avidity: unlike monovalent ligands, nanoscale constructs can simultaneously bind multiple adjacent receptors to augment uptake/selectivity. (c-d) Enhanced stability: 70% of new active pharmaceutical ingredients fail in development because of poor solubility; nanoscale carriers can enhance the circulatory half lives, biodistribution, and intracellular penetration of water-insoluble chemotherapeutics and proteins/nucleic acids (e.g. siRNA) which are susceptible to enzymatic degradation and subject to poor intracellular penetration rates. Scale bars represent 100 μm. Adapted with permission from (a) Ref. , (b) Ref. , (c) , and (d) Ref. . Copyright (a) 2009 Macmillan Publishers Ltd: British Journal of Cancer, (c) 2008 Macmillan Publishers Ltd: Nature Reviews Drug Discovery, (c) 2009 American Chemical Society, and (d) 2010 National Academy of Sciences.

Figure 7. Antiestrogen gold nanoparticles for hormone receptor-targeted breast cancer treatment strategies

(a) Illustration of 25 nm gold nanospheres conjugated with mixed self-assembled monolayers (SAMs) of a high molecular weight polymer stabilizer and a polymer-linked estrogen receptor antagonist, tamoxifen (TAM). Here, TAM acts as a combined targeting and therapeutic moiety which binds membrane- and cytoplasmic-estrogen receptor, inducing apoptosis. (b) High-density gold nanoparticle ligation dramatically accelerates drug influx rates via endocytosis of (10⁴ TAM molecules per particle) versus passive diffusion of the free drug. (c) Optical dark-field scattering microscopy shows nanoparticle uptake into breast cancer cells in a receptor- and ligand-dependent manner. (d) Time-dependent dose-response kinetics show >10⁴ enhanced drug potency from the targeted nanoparticle construct with up to 2.7-fold enhanced potency per equivalent tamoxifen molecule. Adapted with permission from Refs. ^,. Copyright 2009 American Chemical Society and 2011 Royal Society of Chemistry.

Figure 9. Intrinsic pharmacological properties of gold and silver nanoparticles can be leveraged to selectively treat malignant cells

(a) Optical dark-field scattering videography shows that silver (middle panel) and gold (lower panel) nanoparticles targeted with cell-penetrating RGD and nuclear localization sequence (NLS) peptides can selectively impair cell division and induce cytokinesis arrest in malignant cell lines. Confocal immunofluorescence microscopy of (b) control and (c) RGD-targeted gold nanoparticle-treated carcinoma cell nuclei (blue) showed no indication of DNA damage (green) while (d) combined RGD/NLS-targeted gold nanoparticle-treated carcinoma cells showed substantial DNA double strand breaks. (e) Schematic diagram of the proposed sequence of events leading to cellular clustering and non-professional phagocytosis in cancer cells treated with RGD/NLS-targeted silver nanoparticles which generate reactive oxygen species (ROS) that causes DNA damage, programmed cell death, and intercellular signal transduction. (a) Adapted with permission from (a-d) Ref. and (a, e) Ref. Copyright 2010-2011 American Chemical Society.