Selective targeting and thermal destruction of live cells using antibody functionalised gold nanoparticles

University of Technology, Sydney. Faculty of Science.Precious metal nanoparticles have attracted considerable interest on account of their actual or potential applications in chemical, biological or medical analyses, and for their applications in various new types of optical devices or systems. These particles can be engineered to absorb light at a particular wavelength and they can also be chemically functionalised to bind to target cells. Active targeting of the gold particles to the site of a disease can be achieved, in principle, by attaching a suitable antibody to the surface of the gold. Localised heating arises when the affected tissue is irradiated with a laser tuned to the plasmon resonance of the nanoparticle because some of the incident laser light is converted to heat in the particle, which then flows out of the nanoparticle into the target cell. This principle is currently being explored overseas as the basis of a novel form of n1edical treatment for cancer. In this thesis, I extend this concept to develop a method for selectively killing different cellular targets. I report how gold nanoparticles, either spherical or rod-shaped, were functionalised with specific antibodies so that they would selectively attach to particular target cells: murine macrophage cells and tachyzoites of the protozoan parasite Toxoplasma gondii. Following this, the cells were exposed to defined wavelengths and low intensities of continuous laser irradiation from a HeNe laser or a solid state diode laser. Cell viability was determined using nucleic stain dye. Exposure of target cells to specific bioconjugated gold nanoparticles resulted in the highest number of cell death compared with other treatments. In addition, another useful result, independent of the actual process of photothermal therapy, is described in this thesis. This involves the attachment of gold nanoparticle-antibody conjugates to Toxoplasma gondii tachyzoites, which clearly reduced their infection of host cells. Therefore, the research described provides both for an exciting and novel possibility for in vivo killing of any type of target cells using photothermal therapy and for a means to decrease host cell invasion by an intracellular parasite in the body

Destruction and control of Toxoplasma gondii tachyzoites using gold nanosphere/antibody conjugates

The targeting and destruction of Toxoplasma gondii (T.gondii) tachyzoites was studied to be achieved with simple antibody-functionalized gold nanospheres. The nanospheres of approximately 20-nm diameter were conjugated to an antibody specified to T.gondii to produce a gold/antibody conjugate. Microscopic imaging and optical properties indicate the presence of 3000 to 3500 gold nanospheres per tachyzoite. There is no significant photothermal destruction of tachyzoites observed in the absence of Au/anti-T.g. and in this case the number of dead tachyzoites did not increase when the laser dose is increased from 900 to 2100J cm-3. The results also show that an irradiation of 1800J cm -3 caused a cell death rate of 13.5%∓3.6%. The percentage of CHO-K1 cells infected by tachyzoites is inhibited when they are incubated with anti-T.G. alone

Therapeutic possibilities of plasmonically heated gold nanoparticles

Nanoparticles of gold, which are in the size range 10-100 nm, undergo a plasmon resonance with light. This is a process whereby the electrons of the gold resonate in response to incoming radiation causing them to both absorb and scatter light. This effect can be harnessed to either destroy tissue by local heating or release payload molecules of therapeutic importance. Gold nanoparticles can also be conjugated to biologically active moieties, providing possibilities for targeting to particular tissues. Here, we review the progress made in the exploitation of the plasmon resonance of gold nanoparticles in photo-thermal therapeutic medicine. © 2005 Elsevier Ltd. All rights reserved

Optical readout of the intracellular environment using nanoparticle transducers

© 2014 Published by Elsevier Ltd. There is rapid growth in the use of multi-functional nanoparticles as transducers to probe the intracellular environment. New designs of nanoparticles can provide quantitative information at sub-cellular resolution on parameters such as pH, temperature and concentration of nicotinamide adenine dinucleotide (NADH) or selected metal ions. This new work builds on the existing practice of using nanoparticles and fluorescent dyes to provide enhanced microscopic images of cells, but goes beyond it by adding new functionalities and analytical capabilities. In this review, we discuss the recent literature on the development of such nanoparticles for simultaneous biosensing and imaging. We explore and examine the different measurements that will be possible, and analyze the likely accuracy and resolution that could be achieved

Single and multiple detections of foodborne pathogens by gold nanoparticle assays.

A late detection of pathogenic microorganisms in food and drinking water has a high potential to cause adverse health impacts in those who have ingested the pathogens. For this reason there is intense interest in developing precise, rapid and sensitive assays that can detect multiple foodborne pathogens. Such assays would be valuable components in the campaign to minimize foodborne illness. Here, we discuss the emerging types of assays based on gold nanoparticles (GNPs) for rapidly diagnosing single or multiple foodborne pathogen infections. Colorimetric and lateral flow assays based on GNPs may be read by the human eye. Refractometric sensors based on a shift in the position of a plasmon resonance absorption peak can be read by the new generation of inexpensive optical spectrometers. Surface-enhanced Raman spectroscopy and the quartz microbalance require slightly more sophisticated equipment but can be very sensitive. A wide range of electrochemical techniques are also under development. Given the range of options provided by GNPs, we confidently expect that some, or all, of these technologies will eventually enter routine use for detecting pathogens in food. This article is categorized under: Diagnostic Tools > Biosensing

Facile one-pot synthesis of amoxicillin-coated gold nanoparticles and their antimicrobial activity

Nanomaterials have been the object of intense study due to promising applications in a number of different disciplines. In particular, medicine and biology have seen the potential of these novel materials with their nanoscale properties for use in diverse areas such as imaging, sensing and drug vectorisation. Gold nanoparticles (GNPs) are considered a very useful platform to create a valid and efficient drug delivery/carrier system due to their facile and well-studied synthesis, easy surface functionalization and biocompatibility. In the present study, stable antibiotic conjugated GNPs were synthesised by a one-step reaction using a poorly water soluble antibiotic, amoxicillin. Amoxicillin, a member of the penicillin family, reduces the chloroauric acid to form nanoparticles and at the same time coats them to afford the functionalised nanomaterial. A range of techniques including UV-vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) were used to ascertain the gold/drug molar ratio and the optimum temperature for synthesis of uniform monodisperse particles in the ca. 30-40 nm size range. Amoxicillin-conjugated gold showed an enhancement of antibacterial activity against Escherichia coli compared to the antibiotic alone

Quantitative comparison of optimized nanorods, nanoshells and hollow nanospheres for photothermal therapy

The purpose of this study is to get more efficient gold nanoparticles, for necrosis of cancer cells, in photothermal therapy. Therefore a numerical maximization of the absorption efficiency of a set of nanoparticles (nanorod, nanoshell and hollow nanosphere) is proposed, assuming that all the absorbed light is converted to heat. Two therapeutic cases (shallow and deep cancer) are considered. The numerical tools used in this study are the full Mie theory, the discrete dipole approximation and the particle swarm optimization. The optimization leads to an improved efficiency of the nanoparticles compared with previous studies. For the shallow cancer therapy, the hollow nanosphere seems to be more efficient than the other nanoparticles, whereas the hollow nanosphere and nanorod, offer comparable absorption efficiencies, for deep cancer therapy. Finally, a study of tolerance for the size parameters to guarantee an absorption efficiency threshold is included

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging

Gold Nanoparticles Generated in Ethosome Bilayers, As Revealed by Cryo-Electron-Tomography

Gold nanoparticles have been synthesized inside ethosomes, vesicles composed of phospholipid, ethanol and water, which could be very efficient not only in delivery probes to the skin but also as diagnostic and therapeutic multimodal agents. High efficiency encapsulation of gold nanoparticles is achieved by a simple strategy: the nanoparticles synthesis occurs simultaneously with the ethosomes formation, in the absence of any undesirable reducing agents. A three-dimensional reconstruction of a gold-embedded ethosome generated by cryoelectron tomography reveals that the gold particle is localized inside the lipid bilayer, leaving the ethosome surface and core free for further functionalization. The resulting gold nanoparticles are homogeneous in size and shape and, depending on synthesis temperature, the size ranges from 10 to 20 nm, as revealed by TEM. The ethosome-nanoparticles hybrids size has been investigated by means of dynamic light scattering and has been found to vary with temperature and gold salt concentration from 700 to 400 nm. Gold nanoparticles encapsulated ethosomes offer a versatile platform for the enhancement of pharmacological efficacy in transdermal and dermal delivery systems.Comment: 2 videos of the cryo-electron tomographic reconstruction in Supporting Informatio