A comparative analysis of EGFR-targeting antibodies for gold nanoparticle CT imaging of lung cancer.

Computed tomography (CT) is the standard imaging test used for the screening and assessment of suspected lung cancer, but distinguishing malignant from benign nodules by CT is an ongoing challenge. Consequently, a large number of avoidable invasive procedures are performed on patients with benign nodules in order to exclude malignancy. Improving cancer discrimination by non-invasive imaging could reduce the need for invasive diagnostics. In this work we focus on developing a gold nanoparticle contrast agent that targets the epidermal growth factor receptor (EGFR), which is expressed on the cell surface of most lung adenocarcinomas. Three different contrast agents were compared for their tumor targeting effectiveness: non-targeted nanoparticles, nanoparticles conjugated with full-sized anti-EGFR antibodies (cetuximab), and nanoparticles conjugated with a single-domain llama-derived anti-EGFR antibody, which is smaller than the cetuximab, but has a lower binding affinity. Nanoparticle targeting effectiveness was evaluated in vitro by EGFR-binding assays and in cell culture with A431 cells, which highly express EGFR. In vivo CT imaging performance was evaluated in both C57BL/6 mice and in nude mice with A431 subcutaneous tumors. The cetuximab nanoparticles had a significantly shorter blood residence time than either the non-targeted or the single-domain antibody nanoparticles. All of the nanoparticle contrast agents demonstrated tumor accumulation; however, the cetuximab-targeted group had significantly higher tumor gold accumulation than the other two groups, which were statistically indistinguishable from one another. In this study we found that the relative binding affinity of the targeting ligands had more of an effect on tumor accumulation than the circulation half life of the nanoparticles. This study provides useful insight into targeted nanoparticle design and demonstrates that nanoparticle contrast agents can be used to detect tumor receptor overexpression. Combining receptor status data with traditional imaging characteristics has the potential for better differentiation of malignant lung tumors from benign lesions.

Dual-Energy CT Imaging of Tumor Liposome Delivery After Gold Nanoparticle-Augmented Radiation Therapy.

Gold nanoparticles (AuNPs) are emerging as promising agents for both cancer therapy and computed tomography (CT) imaging. AuNPs absorb x-rays and subsequently release low-energy, short-range photoelectrons during external beam radiation therapy (RT), increasing the local radiation dose. When AuNPs are near tumor vasculature, the additional radiation dose can lead to increased vascular permeability. This work focuses on understanding how tumor vascular permeability is influenced by AuNP-augmented RT, and how this effect can be used to improve the delivery of nanoparticle chemotherapeutics. Methods: Dual-energy CT was used to quantify the accumulation of both liposomal iodine and AuNPs in tumors following AuNP-augmented RT in a mouse model of primary soft tissue sarcoma. Mice were injected with non-targeted AuNPs, RGD-functionalized AuNPs (vascular targeting), or no AuNPs, after which they were treated with varying doses of RT. The mice were injected with either liposomal iodine (for the imaging study) or liposomal doxorubicin (for the treatment study) 24 hours after RT. Increased tumor liposome accumulation was assessed by dual-energy CT (iodine) or by tracking tumor treatment response (doxorubicin). Results: A significant increase in vascular permeability was observed for all groups after 20 Gy RT, for the targeted and non-targeted AuNP groups after 10 Gy RT, and for the vascular-targeted AuNP group after 5 Gy RT. Combining targeted AuNPs with 5 Gy RT and liposomal doxorubicin led to a significant tumor growth delay (tumor doubling time ~ 8 days) compared to AuNP-augmented RT or chemotherapy alone (tumor doubling time ~3-4 days). Conclusions: The addition of vascular-targeted AuNPs significantly improved the treatment effect of liposomal doxorubicin after RT, consistent with the increased liposome accumulation observed in tumors in the imaging study. Using this approach with a liposomal drug delivery system can increase specific tumor delivery of chemotherapeutics, which has the potential to significantly improve tumor response and reduce the side effects of both RT and chemotherapy.

In vivo small animal micro-CT using nanoparticle contrast agents.

Computed tomography (CT) is one of the most valuable modalities for in vivo imaging because it is fast, high-resolution, cost-effective, and non-invasive. Moreover, CT is heavily used not only in the clinic (for both diagnostics and treatment planning) but also in preclinical research as micro-CT. Although CT is inherently effective for lung and bone imaging, soft tissue imaging requires the use of contrast agents. For small animal micro-CT, nanoparticle contrast agents are used in order to avoid rapid renal clearance. A variety of nanoparticles have been used for micro-CT imaging, but the majority of research has focused on the use of iodine-containing nanoparticles and gold nanoparticles. Both nanoparticle types can act as highly effective blood pool contrast agents or can be targeted using a wide variety of targeting mechanisms. CT imaging can be further enhanced by adding spectral capabilities to separate multiple co-injected nanoparticles in vivo. Spectral CT, using both energy-integrating and energy-resolving detectors, has been used with multiple contrast agents to enable functional and molecular imaging. This review focuses on new developments for in vivo small animal micro-CT using novel nanoparticle probes applied in preclinical research.

A Plasmonic Gold Nanostar Theranostic Probe for In Vivo Tumor Imaging and Photothermal Therapy.

Nanomedicine has attracted increasing attention in recent years, because it offers great promise to provide personalized diagnostics and therapy with improved treatment efficacy and specificity. In this study, we developed a gold nanostar (GNS) probe for multi-modality theranostics including surface-enhanced Raman scattering (SERS) detection, x-ray computed tomography (CT), two-photon luminescence (TPL) imaging, and photothermal therapy (PTT). We performed radiolabeling, as well as CT and optical imaging, to investigate the GNS probe's biodistribution and intratumoral uptake at both macroscopic and microscopic scales. We also characterized the performance of the GNS nanoprobe for in vitro photothermal heating and in vivo photothermal ablation of primary sarcomas in mice. The results showed that 30-nm GNS have higher tumor uptake, as well as deeper penetration into tumor interstitial space compared to 60-nm GNS. In addition, we found that a higher injection dose of GNS can increase the percentage of tumor uptake. We also demonstrated the GNS probe's superior photothermal conversion efficiency with a highly concentrated heating effect due to a tip-enhanced plasmonic effect. In vivo photothermal therapy with a near-infrared (NIR) laser under the maximum permissible exposure (MPE) led to ablation of aggressive tumors containing GNS, but had no effect in the absence of GNS. This multifunctional GNS probe has the potential to be used for in vivo biosensing, preoperative CT imaging, intraoperative detection with optical methods (SERS and TPL), as well as image-guided photothermal therapy.

Anatomical and functional imaging of myocardial infarction in mice using micro-CT and eXIA 160 contrast agent.

Noninvasive small animal imaging techniques are essential for evaluation of cardiac disease and potential therapeutics. A novel preclinical iodinated contrast agent called eXIA 160 has recently been developed, which has been evaluated for micro-CT cardiac imaging. eXIA 160 creates strong contrast between blood and tissue immediately after its injection and is subsequently taken up by the myocardium and other metabolically active tissues over time. We focus on these properties of eXIA and show its use in imaging myocardial infarction in mice. Five C57BL/6 mice were imaged ~2 weeks after left anterior descending coronary artery ligation. Six C57BL/6 mice were used as controls. Immediately after injection of eXIA 160, an enhancement difference between blood and myocardium of ~340 HU enabled cardiac function estimation via 4D micro-CT scanning with retrospective gating. Four hours post-injection, the healthy perfused myocardium had a contrast difference of ~140 HU relative to blood while the infarcted myocardium showed no enhancement. These differences allowed quantification of infarct size via dual-energy micro-CT. In vivo micro-SPECT imaging and ex vivo triphenyl tetrazolium chloride (TTC) staining provided validation for the micro-CT findings. Root mean squared error of infarct measurements was 2.7% between micro-CT and SPECT, and 4.7% between micro-CT and TTC. Thus, micro-CT with eXIA 160 can be used to provide both morphological and functional data for preclinical studies evaluating myocardial infarction and potential therapies. Further studies are warranted to study the potential use of eXIA 160 as a CT molecular imaging tool for other metabolically active tissues in the mouse.

Dual-energy micro-CT functional imaging of primary lung cancer in mice using gold and iodine nanoparticle contrast agents: a validation study.

PURPOSE: To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. Ex vivo validation studies were performed to demonstrate the accuracy of in vivo contrast agent quantification by CT. METHODS: Primary lung tumors were generated in LSL-Kras(G12D); p53(FL/FL) mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed-two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues. RESULTS: Dual-energy CT enabled in vivo separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R(2) = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with ex vivo measurements. CONCLUSIONS: Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by ex vivo methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT.

Metallization of branched DNA origami for nanoelectronic circuit fabrication.

This work examines the metallization of folded DNA, known as DNA origami, as an enabling step toward the use of such DNA as templates for nanoelectronic circuits. DNA origami, a simple and robust method for creating a wide variety of shapes and patterns, makes possible the increased complexity and flexibility needed for both the design and assembly of useful circuit templates. In addition, selective metallization of the DNA template is essential for circuit fabrication. Metallization of DNA origami presents several challenges over and above those associated with the metallization of other DNA templates such as λ-DNA. These challenges include (1) the stability of the origami in the processes used for metallization, (2) the enhanced selectivity required to metallize small origami structures, (3) the increased difficulty of adhering small structures to the surface so that they will not be removed when subject to multiple metallization steps, and (4) the influence of excess staple strands present with the origami. This paper describes our efforts to understand and address these challenges. Specifically, the influence of experimental conditions on template stability and on the selectivity of metal deposition was investigated for small DNA origami templates. These templates were seeded with Ag and then plated with Au via an electroless deposition process. Both staple strand concentration and the concentration of ions in solution were found to have a significant impact. Selective continuous metal deposition was achieved, with an average metallized height as small as 32 nm. The shape of branched origami was also retained after metallization. These results represent important progress toward the realization of DNA-templated nanocircuits.

Polymerase chain reaction based scaffold preparation for the production of thin, branched DNA origami nanostructures of arbitrary sizes.

Designs for DNA origami have previously been limited by the size of the available single-stranded genomes for scaffolds. Here we present a straightforward method for the production of scaffold strands having various lengths, using polymerase chain reaction amplification followed by strand separation via streptavidin-coated magnetic beads. We have applied this approach in assembling several distinct DNA nanostructures that have thin ( approximately 10 nm) features and branching points, making them potentially useful templates for nanowires in complex electronic circuitry.