Biocompatible Gold Nanorod Conjugates for Preclinical Biomedical Research

Gold nanorods with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide on the surface of the nanorods with 16-mercaptohexadecanoic acid and metoxy-poly(ethylene glycol)-thiol, and the monoclonal antibody HER2. The physical chemistry properties of the conjugates were monitored through optical and zeta-potential measurements to confirm surface chemistry changes. The efficiency of the modifications was quantified through measurement of the average number of antibodies per gold nanorod. The conjugates were investigated for different cells lines: BT-474, MCF7, MCF10, MDCK, and fibroblast. The results show successful cell accumulation of the gold nanorod HER2 conjugates in cells with HER2 overexpression. Incubation of the complexes in heparinized mouse blood demonstrated the low aggregation of the metallic particles through stability of the spectral properties, as verified by UV/VIS spectrometry. Cytotoxicity analysis with LDH release and MTT assay confirms strong targeting and retention of functional activity of the antibody after their conjugation with gold nanorods. Silver staining confirms efficient specific binding to BT-474 cells even in cases where the nanorod complexes were incubated in heparinized mouse blood. This is confirmed through in vivo studies where, following intravenous injection of gold nanorod complexes, silver staining reveals noticeably higher rates of specific binding in mouse tumors than in healthy liver. The conjugates are reproducible, have strong molecular targeting capabilities, have long term stability in vivo and can be used in pre-clinical applications. The conjugates can also be used for molecular and optoacoustic imaging, quantitative sensing of biological substrates, and photothermal therapy

Enabling in vivo measurements of nanoparticle concentrations with three-dimensional optoacoustic tomography

In this report, we demonstrate the feasibility of using optoacoustic tomography (OAT) to evaluate biodistributions of nanoparticles in animal models. The redistribution of single-walled carbon nanotubes (SWCNTs) was visualized in living mice. Nanoparticle concentrations in harvested organs were measured spectroscopically using the intrinsic optical absorption and fluorescence of SWCNTs. Observed increases in optoacoustic signal brightness in tissues were compared with increases in optical absorption coefficients caused by SWCNT accumulation. The methodology presented in this report can further be extended to calibrate the sensitivity of an optoacoustic imaging system for a range of changes in optical absorption coefficient values at specific locations or organs in a mouse body to enable noninvasive measurements of nanoparticle concentrations in vivo. Additionally, qualitative information provided by OAT and quantitative information obtained ex vivo may provide valuable feedback for advancing methods of quantitative analysis with OAT

Protective role of L-methionine against free radical damage of rat brain synaptosomes

Incubation of rat brain synaptosomal/mitochondrial fraction with tert-butylhydroperoxide resulted in accumulation of the lipid peroxidation product, conjugated dienes, damage of the synaptosomal membrane as evidenced by leakage of lactate dehydrogenase, and decrease of the total content of glutathione and of the GSH/GSSG ratio. This treatment also produced a considerable decrease of the ouabain-sensitive ATPase activity and a much smaller diminution of the activities of glutathione reductase and glutathione transferase. Preincubation of the synaptosomal/mitochondrial fraction with 0.5 or 1.0 mM L-methionine significantly protected against lipid peroxidation, membrane damage and changes in the glutathione system produced by low (1 mM) concentrations of tert-butylhydroperoxide and completely prevented inactivation of ouabain-sensitive ATPase, glutathione reductase and glutathione transferase by such treatment. The importance of L-methionine in antioxidant protection is discussed

Temperature-dependent optoacoustic response and transient through zero Grüneisen parameter in optically contrasted media

Non-invasive optoacoustic mapping of temperature in tissues with low blood content can be enabled by administering external contrast agents. Some important clinical applications of such approach include temperature mapping during thermal therapies in a prostate or a mammary gland. However, the technique would require a calibration that establishes functional relationship between the measured normalized optoacoustic response and local tissue temperature. In this work, we investigate how a key calibration parameter – the temperature of zero optoacoustic response (T0) – behaves in different environments simulating biological tissues augmented with either dissolved or particulate (nanoparticles) contrast agents. The observed behavior of T0 in ionic and molecular solutions suggests that in-vivo temperature mapping is feasible for contrast agents of this type, but requires knowledge of local concentrations. Oppositely, particulate contrast agents (plasmonic or carbon nanoparticles) demonstrated concentration-independent thermal behavior of optoacoustic response with T0 defined by the thermoelastic properties of the local environment

Laser nanothermolysis of human leukemia cells using functionalized plasmonic nanoparticles

In the present work, we present the use of gold nanorods as plasmonic nanoparticles for selective photothermal therapy of human acute (HL-60) and chronicle (K-562) leukemia cells using a near-infrared laser. We improved a published methodology of gold nanorods conjugation to generate high yields of narrow band gold nanorods with an optical absorption centered at 760 nm. The manufactured nanorods were pegylated and conjugated with monoclonal antibody to become non-toxic as biocompatible nanothermolysis agent. Gold nanorods are synthesized and conjugated to CD33 monoclonal antibody. After pegylation, or conjugation with CD33 antibody, gold nanorods were non-toxic to acute and chronic leukemia cells. Our modified gold nanorods CD33 conjugates shown high level of accumulation for both leukemia cell lines, and successful used for nanothermolysis of human leukemia cells in vitro. Each sample was illuminated with 1 or 3 laser shots as for low and for high laser fluence. The radiation was provided by a Quanta Systems q-switched titanium sapphire laser, and the system was designed for maximum sample coverage using non-focused illumination. HL-60 and K-562 cells were treated for 45 min with gold nanorods CD33 conjugated, or with pegylated gold nanorods. The effect of pulsed-laser nanothermolysis for acute and chronic leukemia cells were investigated with cell counting for number of living cells, percentage of cell death and functional parameters such as damage of cell membrane and metabolic activity. Gold nanorods CD33 conjugates significantly increase cell damage for low fluence laser and completely destroyed cancer cells after 3 pulses for low fluence (acute leukemia) and for high fluence laser as for HL-60 (acute) and for K-562 (chronicle) leukemia cells

Biocompatible Gold Nanorod Conjugates for Preclinical Biomedical Research

Gold nanorods with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide on the surface of the nanorods with 16-mercaptohexadecanoic acid and metoxy-poly(ethylene glycol)-thiol, and the monoclonal antibody HER2. The physical chemistry properties of the conjugates were monitored through optical and zeta-potential measurements to confirm surface chemistry changes. The efficiency of the modifications was quantified through measurement of the average number of antibodies per gold nanorod. The conjugates were investigated for different cells lines: BT-474, MCF7, MCF10, MDCK, and fibroblast. The results show successful cell accumulation of the gold nanorod HER2 conjugates in cells with HER2 overexpression. Incubation of the complexes in heparinized mouse blood demonstrated the low aggregation of the metallic particles through stability of the spectral properties, as verified by UV/VIS spectrometry. Cytotoxicity analysis with LDH release and MTT assay confirms strong targeting and retention of functional activity of the antibody after their conjugation with gold nanorods. Silver staining confirms efficient specific binding to BT-474 cells even in cases where the nanorod complexes were incubated in heparinized mouse blood. This is confirmed through in vivo studies where, following intravenous injection of gold nanorod complexes, silver staining reveals noticeably higher rates of specific binding in mouse tumors than in healthy liver. The conjugates are reproducible, have strong molecular targeting capabilities, have long term stability in vivo and can be used in pre-clinical applications. The conjugates can also be used for molecular and optoacoustic imaging, quantitative sensing of biological substrates, and photothermal therapy