«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
It has been reported that the optical properties of nanoparticle (i.e. the optical resonance wavelength, the extinction cross-section, and the relative contribution of scattering to the extinction) are strongly dependent on the nanoparticle dimensions, allowing tunability for specific applications . The increase in the nanoparticle size results in an increase in the extinction as well as the relative contribution of scattering.
This phenomenon is also described by Yguerabide et al. by the following equation .
Where Csca is light scattering cross section, a is particle radius, nmed is the refractive index of the medium surrounding the particle, m is the relative refractive index of the bulk particle material, and λ0 is the wavelength of the incident beam.
As seen in Equation 2-2, the scattering cross-section increases by 6 power of the particle radius in which increase in particle radius results in higher cross-section for stronger light scattering. Furthermore, gold nanoparticles provide high-scattering crosssection compared to the conventionally used fluorescent dyes by 4-5 orders in magnitude . Thus, surface plasmon oscillation of electrons of the gold nanoparticle result in strongly enhanced scattering that is useful for various biomedical applications.
Advantages in Using Gold Nanoparticle Size and Shape Tunability Gold nanoparticles are easily synthesized in various shapes and sizes that it confers size and shape tunability. In general, metal salts are reduced by reducing agents in a controlled manner to produce spherical nanoparticles. The ratio between the metal salt and the reducing agent determine the size of the nanoparticle. Some of the frequently used methods to synthesize spherical gold nanoparticles are 1) the Turkevich method (1951) that reduces the gold chloride by citrate in boiling water, 2) the related Frens method (1973), 3) the Brust method (1994) for smaller (~2nm) gold nanoparticles, where aqueous gold ion solution is transferred to an organic phase, mediated by phase transfer agent, followed by reduction with borohydride, 4) microemulsion method, and 5) seeding method in which gold seed particles are used to grow more gold in the presence of of a weak reducing agent . Spherical shapes are the easiest to synthesize since spheres are the lowest-energy shape.
Gold nanorods are synthesized in various methods such as seed-mediated growth methods [81, 85]. Nanoshells are synthesized by having spherical dielectric nanoparticle (i.e. silica nanoparticle) surrounded by an ultrathin, conductive, metallic layer (i.e. gold) . Most recently, Xia group of the University of Washington have created gold nanocages sizes ranging from 10 to 150nm, which are porous gold nanoparticles. Gold nanocages are created by reacting silver nanoparticles with chloroauric acid in boiling water .
TEM Images of Various Shapes and Sizes of Gold Nanoparticles: a-d represent spherical gold nanoparticle, f-k represent gold nanrods, l represents gold nanocage, and m represent gold nanoshells [a-j: ; l: ; m: ].
Biocompatibility Gold nanoparticles are inert and have low in vivo toxicity compared to the other metallic materials [93, 94]. Several groups have examined the cellular toxicity of gold nanoparticles (Table 2.1). It was found that gold nanoparticles show little or no cytotoxicity in several studies. The biocompatibility of gold nanoparticle suggests that biological effect of gold nanoparticle is unlikely due to the intrinsic toxicity of the metal.
Summary of Selected Cytotoxicity for Gold Nanoparticles  Easy Detection by Using Various Analytical Methods Ultraviolet-Visible Spectroscopy The absorption spectra for spherical nanoparticle depend directly on the size of the nanoparticle (i.e. extrinsic size effect) due to the surface plasmon resonance .
Particularly for gold nanoparticles, they have a strong visible-light plasmon resonance that Ultraviolet-Visible (UV-Vis) Spectroscopy is useful for characterization. As the particle size gets larger, we can observe red shift and increase in bandwidth in the absorption spectra obtained from the UV-Vis Spectroscopy (Figure 2.5). Red shift (and/or broadening of the bandwidth of the absorption peak) is also observed when the nanoparticle is coated with different ligands or aggregation occurs within the gold nanoparticles in the solution. Thus, UV-Vis Spectroscopy is also useful to check the stability of the colloidal gold in solution.
Increase in Particle size results in red shift and increased bandwidth in absorption spectra  The Beer-Lambert law can be used to calculate the concentration of the solution.
Where A is the measures absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength through the sample, c is the concentration of the absorbing species, and ε is a constant known as molar absorptivity or extinction coefficient.
The Beer-Lambert law states that the absorbance of the solution is directly proportional to the concentration of the absorbing species and the path length. Thus, for a fixed path length with known extinction coefficient, UV-Vis Spectroscopy can be used to determine the concentration of the absorbing species in the solution.
Darkfield Light-Scattering Imaging Colloidal gold nanoparticle has become an important alternative as imaging agents due to their biocompatibility and nonsusceptibility to photo-bleaching or chemical/thermal denaturation, a common problem observed with organic dyes .
Recently, El-Sayed et al. demonstrated differentiation of cancerous cells from healthy cells by darkfield light –scattering imaging with 35nm gold nanoparticle .
Figure 2.6 Darkfield Light-Scattering Images of HSC Cancer Cell: HSC cancerous cells without (left column) gold nanoparticles and HSC cancerous cells with (right column) gold nanoparticles that scatter light to display bright yellow glow (scale bar: 10 μm)  Darkfield light-scattering imaging utilizes microscopy techniques.
The darkfield imaging microscopy requires a condenser that has numerical aperture higher than the objective. The condenser delivers a very narrow beam of white light from the light source. Then the objective collects the only scattered light (not transmitted light from the samples) that the center of the illuminating beam is blocked from the entering light collection cone of the microscope objective and only the scattered light of the side beam is collected. As a result, a bright image with a dark background is created (Figure 2.7).
Diagram of Darkfield Light-Scattering Microscopy Setup A high-scattering cross-section is essential for imaging applications based on darkfield light-scattering microscopy. Gold nanoparticles provide cross-sections of 4-5 orders higher in magnitude than that of the conventional organic dyes. Darkfield allows label-free detection that does not require staining of the sample. It also creates a distinctive image that reflects the true-color of the gold nanoparticle, which depends on the size and shape of the particle.
Fluorescence Resonant energy-transfer is observed in fluorescent ligand-capped gold nanoparticles. For most of chemisorbed chromophores on gold surface, quenching of the fluorescence is observed. Quenching is generally due to increased non-radiative relaxation of the excited state due to energy and/or electron transfer . Also, quenching is partly due to a decrease in the rate of radiative relaxation related to changes in the photonic mode density near the metal cluster surface (plasmonic effect) . Both radiative and non-radiative rates critically depend on the size and shape of the nanoparticle, the distance between the dye molecules, the orientation of the dipole with respect to the dye-nanoparticle axis, and the overlap of the molecule’s emission with the nanoparticle’s absorption spectrum . Also, enhancement of fluorescence by metal nanoparticles is reported, mostly occurring in aggregated metal colloids .
Fluorescence Quenching of Chemisorbed Chromophore on Gold Nanoparticle  Fluorescence quenching phenomenon can be used to check successful ligand exchange process. When thiolated chromophore, such as doxorubicin, is added to the gold colloid solution, the fluorescence of doxorubicin is quenched by successful coating or chemisorption of thiolated doxorubicin onto gold nanoparticle surface, where quenching is mediated by the thiol group.
Transmission Electron Microscopy High Resolution Transmission electron microscopy (TEM) is the most common characterization technique used to photograph the gold core of the gold nanoparticle.
TEM can be used to verify the morphology and size of the gold nanoparticle. Due to the electro-dense surface of the gold nanoparticle, gold core is visible as dark spots in the TEM images.
Surface Enhanced Raman Scattering (SERS) SERS is a spectroscopic technique that results from strongly increased Raman signals when molecules are attached to nanometer-sized gold nanostructures. Gold nanoparticle has unique optical properties that Raman signal from adsorbed reporter molecules can be increased up to 1014~1015 orders in magnitude, allowing “single” molecular level spectroscopic detection through SERS [102-104]. SERS, an analytical technique, can give information on any small chemical changes occurring at the surface and interfaces of gold nanoparticle [103, 105].
SERS has generated considerable interest, particularly in molecular diagnosis and in vivo Raman spectroscopy and imaging. SERS nanoparticle tags, gold nanoparticle with embedded reporter molecules, are capable of providing detailed spectroscopic information and results in signals that are much brighter than semiconductor quantum dots in near-infrared spectral window . Recently, Qian et al. has demonstrated in vivo spectral imaging of cancer in a mouse model (Figure 2.9) .
In vivo Cancer Targeting and Surface-Enhanced Raman Detection by Using Antibody-Conjugated Gold Nanoparticle: SERS spectra obtained from the tumor and liver locations by using (a) targeted and (b) non-targeted nanoparticles. (c) is pictures showing a laser beam focusing to the tumor site or the liver to obtain SERS spectroscopic signal  Inductive-Coupled Plasma Mass Spectroscopy Due to lack of presence of elemental gold in animals, the major advantage of using gold in biological application is to use it as a “tracer” to quantitatively detect the accumulated gold in various organs or tumor by elemental mass spectroscopy . Gold nanoparticle is generally insoluble and rarely present in the biological tissues that it makes easy to detect even at low concentrations using methods such as inductive coupled plasma-mass spectroscopy (ICP-MS). Unlike the “qualitative” approach to detect the presence/absence of the molecule of interest by immunohistochemical staining, ICP-MS allows to directly “quantify” the exact amount of atom/particle of interest at the location of interest. ICP-MS also allows comparing of relative amount of atom/ particle of interest at different organs in a single or multiple subjects.
Dynamic Light Scattering and Zeta Potential In conjunction with the TEM images, dynamic light scattering (DLS) can be used to characterize the size of the gold nanoparticle. Furthermore, successful ligand exchange process can be verified via dynamic light scattering and zeta potential measurements. As the gold nanoparticle is coated with surface ligands, the hydrodynamic diameter increases with the addition of the surface ligands, seen in the DLS measurements. Similarly, with the addition of surface ligands, the surface charge or the zeta potential of the gold nanoparticle becomes more positive.
Easy Surface Modification The surface of gold nanoparticle can be easily modified via place exchange reaction to introduce new physical and chemical properties. Surface capping ligand can provide 1) functionality (i.e. targeting ligand for cellular detection, delivery of therapeutic or imaging agent) [56, 97, 107], 2) colloidal stability to prevent aggregation , 3) nonfouling surfaces to prevent opsonization by RES (i.e PEGylation) , 4) positively, neutrally, or negatively charged hydrophilic monolayer [109-111], or 5) hydrophobic monolayer [112, 113]. Gold surface is usually coated via thiol-gold bond interactions, where chemisorbed thiol onto gold surface has high bond energy of 47kcal/mol, compared to weak 6 kcal/mol of amine-gold bond, for strong covalent bonding .
Different PEG Configurations on Gold Surface: Low surface coverage of PEG chains lead to mushroom (a) configuration and chains are located closer to the gold surface. A high surface coverage of PEG chain leads to brush (b) configuration that PEG chains are extending away from the surface  The place exchange reaction occurs when clean metal is immersed in a dilute solution of thiols or disulfides to produce well-defined, organized, self-assembled structures at the metal/liquid surface. The place exchange reaction starts with initial rapid (kinetically driven) adsorption of a monolayer, followed by slower processes which results in the formation of the thermodynamically favored layer . It has been speculated that longer thiolated ligands displaces the shorter thiolated ligands, which are bound onto gold surface . Also, the exchange occurs preferentially at minority sites such as defects on the gold surface that 1) the incoming ligand penetrates less crowded site of the monolayer in order to undergo place-exchange or 2) bound ligands undergo desorption (preferentially at the defect sites), followed by incoming thiol attachment to the newly created surface vacancy .
Depending on the concentration of ligands added to the gold colloid solution, various configurations of polymer monolayers can form. The two representative configurations are “mushroom” and “brush” configurations (Figure 2.10). Brush configuration tends to give full surface coverage of the gold surface that result in better colloidal stability in high salt in vivo conditions.