Anton Liopo

Engineering of hetero-functional gold nanorods for the in vivo molecular targeting of breast cancer cells

A novel technique is described to functionalize gold nanorods (GNRs) allowing for in vivo targeting of breast cancer tumors grown in athymic nude mice. GNRs were functionalized by covalent attachment of Herceptin (HER), a monoclonal antibody that enables molecular recognition of breast cancer cells expressing highly specific tumor associated antigens, and poly(ethylene glycol) (PEG) which obscures particles against the reticuloendothelial system in the body. The stability and functionality of fabricated particles (Her-PEG GNRs) were demonstrated in vitro in the presence of blood and then in vivo in nude mice model for breast cancer. The results demonstrate successful tumor accumulation of functionalized gold nanorods within HER2/neu overexpressing breast tumors in tumor-bearing nude mice and support the notions that GNRs can be used for molecular imaging of tumor.

Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo

The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Melanin nanoparticles as a novel contrast agent for optoacoustic tomography.

We describe the synthesis and characterization of melanin-like nanoparticles (MNP) as novel contrast agents for optoacoustic tomography. Good dispersion stability of high concentration MNPs in different biological media was achieved with thiol-terminated methoxy-poly(ethylene glycol), which can be used for further functional conjugation. MNP-PEG were found biocompatible with human MCF-7 and 3T3 cells. Cell toxicity of MNPs was found lower than that of gold nanorods for concentrations that provide equal optical absorbance. Optoacoustic tomography images were obtained with Laser Optoacoustic Imaging System (LOIS-3D) from tubes filled with contrast agents and live mice. Imaging of tubes permitted verification of the system resolution <300 μm and sensitivity Δμa=0.03/cm under safe laser fluence of 20 mJ/cm2. Water suspensions of MNP demonstrated optoacoustic efficiency that is about equal to that of gold nanorods under conditions of equal optical absorption. We conclude that MNPs have the potential for biomedical imaging applications as optoacoustic contrast agents.

Three-dimensional optoacoustic imaging as a new noninvasive technique to study long-term biodistribution of optical contrast agents in small animal models

Abstract. We used a 3-D optoacoustic (OA) tomography system to create maps of optical absorbance of mice tissues contrasted with gold nanorods (GNRs). Nude mice were scanned before and after injection of GNRs at time periods varying from 1 to 192 h. Synthesized GNRs were purified from hexadecyltrimethylammonium bromide and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of purified GNR-PEG complexes resulted in enhanced OA contrast of internal organs and blood vessels compared to the same mouse before injection of the contrast agent. Maximum enhancement of the OA images was observed 24 to 48 h postinjection, followed by a slow clearance trend for the remaining part of the studied period (eight days). We demonstrate that OA imaging with two laser wavelengths can be used for noninvasive, long-term studies of biological distribution of contrast agents.

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.

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.

Optoacoustic 3D whole-body tomography: experiments in nude mice

We developed a 3D whole-body optoacoustic tomography system for applications in preclinical research on mice. The system is capable of generating images with resolution better than 0.6 mm. Two pulsed lasers, an Alexandrite laser operating at 755 nm and a Nd:YAG laser operating at 532 nm and 1064nm were used for light delivery. The tomographic images were obtained while the objects of study (phantoms or mice) were rotated within a sphere outlined by a concave arc-shaped array of 64 piezo-composite transducers. During the scan, the mouse was illuminated orthogonally to the array with two wide beams of light from a bifurcated fiber bundle. Illumination at 532 nm showed superficial vasculature, but limited penetration depth at this wavelength prevented the detection of deeper structures. Illumination at 755 and 1064 nm showed organs and blood vessels, respectively. Filtering of the optoacoustic signals using high frequency enhancing wavelets further emphasized the smaller blood vessels.

PEG-coated gold nanorod monoclonal antibody conjugates in preclinical research with optoacoustic tomography, photothermal therapy, and sensing

Gold nanorods (GNR) 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 (CTAB) on the surface of GNR with 16-mercaptohexadecanoic acid (MHDA) and metoxy-poly(ethylene glycol)-thiol (PEG), and the monoclonal antibodies: HER2 or CD33. The physical chemistry property of the conjugates was monitored through optical and zetapotential measurements to confirm surface chemistry. The plasmon resonance is kept in the near infrared area, and changes from strong positive charge for GNR-CTAB to slightly negative for GNR-PEG-mAb conjugates are observed. The conjugates were investigated for different cells lines: breast cancer cells and human leukemia lines in vivo applications. These results demonstrate successful tumor accumulation of our modified PEG-MHDA conjugates of GNR for HER2/neu in both overexpressed breast tumors in nude mice, and for thermolysis of human leukemia cells in vitro. The conjugates are non-toxic and can be used in pre-clinical applications, as well as molecular and optoacoustic imaging, and quantitative sensing of biological substrates.

Gold nanorod distribution in mouse tissues after intravenous injection monitored with optoacoustic tomography

We used a three-dimensional optical tomogaphy system that was previously developed to create high contrast maps of optical absorbance of mice tissues. In this study, animals were scanned before and after injection of gold nanorods (GNRs) at different time periods. As-synthesized GNRs were purified from hexadecyltrimethylammonium bromide (CTAB) and coated with polyethylene glycol (PEG) to obtain GNR-PEG complexes suitable for in vivo applications. Intravenous administration of the purified GNR-PEG complexes to mice resulted in an enhanced contrast of normal tissues and blood vessels as compared to ordinary nude mice. In parallel with optoacoustic imaging we investigated the accumulation of GNRs in liver using invasive analytical techniques. Maximum levels of GNRs in liver macrophages were observed after 48-72 hours post-injection, followed by slow clearance trend after 8 days. Optoacoustic imaging revealed redistribution of GNR in mouse organ and tissues: in the initial hours, accumulation of GNRs is seen predominantly in the periphery of the mouse, while a gradual increase of GNR levels in liver, spleen and kidneys is seen in 1 and 24 hours.