Off to the organelles - killing cancer cells with targeted gold nanoparticles.

Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment.

Nanoparticle-based caspase sensors.

Recent advances in nanotechnology have provided new tools for measuring enzymatic activities that are relevant for the assessment of physiological and pathological processes. Caspases, the enzymes intimately linked with cell death and inflammation, are cysteine-dependent aspartate-directed proteases. The measurement of caspase activity requires assays that can provide data with specificity, precision and sensitivity. Several nanoparticle-based assays are now beginning to emerge. This article will first provide a brief discussion of conventional methods of measuring caspase activity and their limitations, followed by an overview of the advantages and limitations of nanoparticle-based strategies for sensing caspase enzymatic activity in vitro and in vivo.

Nanoparticle-Based and Bioengineered Probes and Sensors to Detect Physiological and Pathological Biomarkers in Neural Cells.

Nanotechnology, a rapidly evolving field, provides simple and practical tools to investigate the nervous system in health and disease. Among these tools are nanoparticle-based probes and sensors that detect biochemical and physiological properties of neurons and glia, and generate signals proportionate to physical, chemical, and/or electrical changes in these cells. In this context, quantum dots (QDs), carbon-based structures (C-dots, grapheme, and nanodiamonds) and gold nanoparticles are the most commonly used nanostructures. They can detect and measure enzymatic activities of proteases (metalloproteinases, caspases), ions, metabolites, and other biomolecules under physiological or pathological conditions in neural cells. Here, we provide some examples of nanoparticle-based and genetically engineered probes and sensors that are used to reveal changes in protease activities and calcium ion concentrations. Although significant progress in developing these tools has been made for probing neural cells, several challenges remain. We review many common hurdles in sensor development, while highlighting certain advances. In the end, we propose some future directions and ideas for developing practical tools for neural cell investigations, based on the maxim "Measure what is measurable, and make measurable what is not so" (Galileo Galilei).

Gold nanoparticles induce nuclear damage in breast cancer cells, which is further amplified by hyperthermia.

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.

Caspase-1 activity in microglia stimulated by pro-inflammagen nanocrystals.

Although caspase-1 is a key participant in inflammation, there is no sensitive assay to measure its enzymatic activity in real time in cells or animals. Here we describe a nanosensor for caspase-1 ratiometric measurements, consisting of a rhodamine-labeled, caspase-1 cleavable peptide linked to quantum dots (QDs). Microglia cells were stimulated by lipopolysaccharide (LPS) and by hybrid nanoparticles LPS-QDs. These stimuli activated caspase-1 in microglia monolayers and in the mouse brain, while a selected caspase inhibitor markedly reduced it. LPS-QDs entered into the lysosomal compartment and led to an enlargement of these cellular organelles in the exposed microglia. Both lysosomal swelling and mitochondrial impairment contributed to caspase-1 activation and to the consequent interleukin-1ß release. The results from these studies highlight how the unique properties of QDs can be used to create versatile biotools in the study of inflammation in real time in vivo.

Gold-nanoparticle-based biosensors for detection of enzyme activity.

Optimal enzyme activity is essential for maintenance of physiological homeostasis. A variety of both non-genetic and genetic disruptions can excessively activate or silence intrinsic enzyme activities, with pathological outcomes. Many pharmacological agents are activators and inhibitors of enzymes. It is essential, therefore, in the development of drugs as enzyme activators and inhibitors, that enzyme activities be accurately measured under physiological and pathological conditions. Different biochemical assays have been developed for this purpose, some of which are based on nanostructured materials. This review focuses on gold nanoparticle (GNP)-based structures for the sensing and measurement of enzyme activities in biological specimens. Here we provide an overview and critical analysis of such assays, identify their advantages and limitations, and discuss interesting features of GNPs to be exploited for future applications in pharmacology.

Gold nanoparticles and quantum dots for bioimaging.

Nanoparticles are the latest tool acquired by the science of bioimaging, serving primarily as new contrast agents, sensors, or signal enhancing agents in established and developing imaging techniques. This review focuses on the unique properties of two classes of nanoparticles: gold nanoparticles (GNP) and quantum dots, and how these properties are benefiting cellular and in vivo imaging. We discuss the surface plasmon resonance of GNP and its implications for various imaging techniques of biological relevance. Furthermore, the key properties of quantum dots are reviewed, and their use alone or in combination with traditional fluorescent dyes for biological imaging are described. The underlying principles of these techniques are provided, along with some representative examples.

Microglial response to gold nanoparticles.

Given the emergence of nanotherapeutics and nanodiagnostics as key tools in today's medicine, it has become of critical importance to define precisely the interactions of nanomaterials with biological systems and to characterize the resulting cellular response. We report here the interactions of microglia and neurons with gold nanoparticles (GNPs) of three morphologies, spheres, rods, and urchins, coated with poly(ethylene glycol) (PEG) or cetyl trimethylammonium bromide (CTAB). Microglia are the resident immune cells of the brain, primarily involved in surveillance, macrophagy, and production of cytokines and trophic factors. Analysis by dark-field microscopy and by two-photon-induced luminescence (TPL) indicates that the exposure of neural cells to GNPs resulted in (i) GNP internalization by both microglial cells and primary hippocampal neurons, as revealed by dark-field microscopy and by two-photon-induced luminescence (TPL), (ii) transient toll-like receptor 2 (TLR-2) up-regulation in the olfactory bulb, after intranasal administration in transgenic mice, in vivo, in real time, and (iii) differential up-regulation in vitro of TLR-2 together with interleukin 1 alpha (IL-1alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and nitric oxide (NO) in microglia. The study demonstrates that GNP morphology and surface chemistry strongly influence the microglial activation status and suggests that interactions between GNPs and microglia can be differentially regulated by tuning GNP nanogeometry.

Ultrathin molecularly imprinted polymer sensors employing enhanced transmission surface plasmon resonance spectroscopy.

An ultrathin novel nanosensor (31.5 +/- 4.1 nm thick in the absence of analytes), employing a molecularly imprinted polymer as a recognition element for cholesterol and gold nanoparticle enhanced transmission surface plasmon resonance spectroscopy for detection, was constructed.

Nanosensors based on responsive polymer brushes and gold nanoparticle enhanced transmission surface plasmon resonance spectroscopy.

Swelling (and shrinking) of poly(2-vinylpyridine), P2VP, polymer brushes, caused by pH changes, could be readily monitored by transmission surface plasmon resonance, T-SPR, spectroscopy. Gold nanoparticles attached to the P2VP polymer brushes dramatically enhanced the pH-induced shift in the T-SPR absorption spectra. (A 50 nm shift of the absorption maximum of the T-SPR spectrum of the supporting gold nanoislands was observed upon changing the pH from 5.0 to 2.0, corresponding to a swelling of the polymer brushes from 8.1 +/- 0.7 to 24.0 +/- 2.0 nm. Same shift in the opposite direction was observed upon changing the pH from 2.0 to 5.0.)

Hybridization of oligonucleotide-modified silver and gold nanoparticles in aqueous dispersions and on gold films.

Functionalization of silver and gold nanoparticles by 12mer-thiolated homo-oligonucleotides, SA and ST (containing only adenine or thymine, respectively), and their hybridization and dehybridization in aqueous dispersions have been described. In addition, ST and SA were self-assembled onto gold films and hybridized with their complementary pairs, unlabeled or labeled by gold and silver nanoparticles. The base pairing between DNA strands and the types of oligonucleotides (adenine or thymine) attached to the nanoparticles was detected by Polarization Modulated Fourier Transform Infrared Reflection Absorption Spectroscopy (PM-FTIRRAS).

Digital signal processing for step-scan phase and electrochemical potential double-modulation Fourier transform infrared spectrometry.

Step-scan double-modulation (phase and electrochemical potential) Fourier transform infrared (FT-IR) spectrometry provides both spectroscopic and dynamic information about faradaic reactions. Recently introduced digital signal processing (DSP) can be used, instead of two lock-in amplifiers, for the optical signal demodulation at two modulation frequencies. In order to establish the merits of double-modulation FT-IR spectrometry with DSP, spectro-electrochemical experiments are performed in the attenuated total reflection configuration and with the commonly used ferri/ferrocyanide redox couple. Because of a large potential drop associated with the uncompensated resistance, a satisfactory signal-to-noise ratio for the alternating current (ac) optical measurements is obtained only with the employment of positive feedback compensation. In this arrangement, the amplitude of electrochemical modulation is sufficiently large to convert a significant fraction of the reduced form to the oxidized form and back to the reduced form. Large amplitude ac voltammetry demonstrates that the phase of faradaic admittance at the formal potential is approximately 45 degrees at 2.00 Hz. In addition, these experiments allow for calculation of the interfacial ac potential. This variable is needed for the normalization of the in-phase and the quadrature spectra in order to overcome the problem associated with the iR(u) drop. Because of the integral relationship between the faradaic current and the electromodulation reflectance coefficient, the phases of electromodulation reflectance coefficient with respect to the interfacial ac potential are expected to be -45 degrees and 135 degrees for the reduced and oxidized forms, respectively. However, dynamic information from double-modulation FT-IR spectrometry is available only if demodulation at the electrochemical potential modulation frequency is performed with respect to a defined phase. Because of an undefined demodulation phase implemented in the current version of DSP software, step-scan double-modulation FT-IR spectrometry with DSP is suitable only to provide spectroscopic information. In order to overcome this limitation, the demodulation of the ac optical signal at the electrochemical modulation frequency must be synchronized in phase with the ac potential modulation applied to the electrochemical cell.

Size quantized formation and self-assembly of gold encased silver nanoparticles.

Mixing aqueous dispersions of thiocyanate ion coated small (< 3.5 nm diameter) gold nanoparticles and EDTA covered larger (> 22 nm diameter) silver nanoparticles, results in the formation of robust gold encased silver nanoparticles; in contrast to using larger (> 11 nm diameter) gold nanoparticles which forms chained structures.