In-vivo Optical Imaging Research Articles

Fast wavefront shaping for two-photon brain imaging with multipatch correction.

Nonlinear fluorescence microscopy promotes in-vivo optical imaging of cellular structure at diffraction-limited resolution deep inside scattering biological tissues. Active compensation of tissue-induced aberrations and light scattering through adaptive wavefront correction further extends the accessible depth by restoring high resolution at large depth. However, those corrections are only valid over a very limited field of view within the angular memory effect. To overcome this limitation, we introduce an acousto-optic light modulation technique for fluorescence imaging with simultaneous wavefront correction at pixel scan speed. Biaxial wavefront corrections are first learned by adaptive optimization at multiple locations in the image field. During image acquisition, the learned corrections are then switched on the fly according to the position of the excitation focus during the raster scan. The proposed microscope is applied to in vivo transcranial neuron imaging and demonstrates multi-patch correction of thinned skull-induced aberrations and scattering at 40-kHz data acquisition speed.

Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site.Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI.Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI.Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.

In Vivo Assessment of Protein-Protein Interactions Using BRET Assay.

Proteins play an important part in almost all life activities and across all organisms. Proteins occasionally act on their own but rather fulfill most of their biological tasks by cooperating with other proteins or ligand molecules. The bioluminescence resonance energy transfer (BRET) assay serves to measure dynamic events such as protein-protein or protein-ligand interactions in vitro or in-vivo. With several inherent attributes such as rapid and fairly sensitive ratio-metric measurements, assessment of interactions irrespective of protein location within the cellular compartment, cost-effectiveness consenting to high-throughput screening compatibility, makes BRET a popular genetic reporter-based assay system for protein-protein interaction (PPI) studies. Based on the Förster principle, BRET allows to judge if the proximity has been achieved between the interacting partners. In recent years, the BRET application has emerged as a significantly versatile assay format by using multiple detection devices such as a plate reader or in-vivo optical imaging platform, or even a bioluminescence microscope has expanded its scope for advancing PPI studies. Beyond the scope of quantitative measurement of PPIs, molecular optical imaging applications based on BRET assay have expanded the scope for screening pharmacological compounds by unifying live cell and in-vivo animal-/plant-based experiments using the same platform technology. In this chapter, we have given intricate methodological details for performing in-vitro and in-vivo BRET experiments, primarily by using donor/acceptor reporter protein combinations.

Image-guided focused ultrasound-mediated drug delivery andcomparison between 2-D and 3-D therapeutic strategies

The objective of this research was to test a new US image-guided FUS (USgFUS) system for stimulating microvascular permeabilization and enhancing intratumoral drug delivery in volume space. 3-D USgFUS therapy functionality was implemented on a programmable US system with a dual transducer for interleaved imaging and 3-D therapy. 3-D therapy consisted of focal zones distributed over a user-defined volume whereas 2-D treatment had the same focal zones but about a single plane. Using breast cancer-bearing mice (N = 4), US therapy was conducted with a mechanical index of 0.5, pulse repetition frequency of 10 Hz, and duty cycle of 10%. Therapy was performed immediately after a 100 μl bolus injection of microbubbles and IR-780 dye. In-vivo optical imaging was performed at 0, 24, and 48 h. Tumors were then excised for ex-vivo analysis. 3-D USgFUS treatment increased dye extravasation within the tumor space at 24 (245.5%) and 48 h (297.5%) when compared to 2-D US therapy (p = 0.01). Ex vivo optical images revealed a similar trend with 3-D US treatment producing a higher fluorescent signal than the 2-D approach. Preliminary results suggest that 3-D USgFUS-mediated drug delivery is an exciting prospect for treatment of the entire cancer burden.

Influence of europium (Eu) doped tantalum oxide nanoparticles (TaOx NPs): A potential contrast agent

We report a one of a kind study to develop Europium doped Tantalum oxide nanoparticles for XEOL imaging. X-ray excited optical luminescence (XEOL) is an emerging alternative to in-vivo optical imaging to visualize deep tissues. Therefore, a proper selection of host material is essential to generate high luminescent XEOL probes to acquire molecular information about diseases. Herein, Tantalum oxide nanoparticles (TaOx NPs), a well-known biocompatible CT contrast agent, is used as the host material. In this article, we present the experimental investigation of X% of Europium doped into TaOx nanoparticles (Eu: TaOx NPs) as a potential contrast for XEOL imaging. XEOL measured with TaOx and X% Eu: TaOx NPs showed blue and reddish-orange luminescence when exposed to X-rays. By changing the X-ray voltages between 50 and 200 keV, the intensity of reddish-orange luminescence of X% Eu: TaOx NPs increases gradually. Based on the results, we propose that the enhanced reddish-orange luminescence from Eu: TaOx NPs is due to the energy transfer from host material and direct radiative decay from luminescent centre.

Residual thrombus following plaque disruption contributes to rapid plaque progression: in-vivo serial optical coherence tomography imaging

Residual thrombus following plaque disruption contributes to rapid plaque progression: in-vivo serial optical coherence tomography imaging

Time-Multiplexed Illumination for simultaneous Structural and Functional Voltage Sensitive Dye Recordings with a single Photo Sensor.

The in-vivo optical imaging of the cortical surface provides the ability to record different types of biophysiological signals, e.g., structural information, intrinsic signals, like blood oxygenation coupled reflection changes as well as extrinsic properties of voltage sensitive probes, like fluorescent voltage-sensitive dyes. The recorded data sets have very high temporal and spatial resolutions on a meso- to macroscopic scale, which surpass conventional multi-electrode recordings. Both, intrinsic and functional data sets, each provide unique information about temporal and spatial dynamics of cortical functioning, yet have individual drawbacks. To optimize the informational value it would thus be opportune to combine different types of optical imaging in a near simultaneous recording.Due to the low signal-to-noise ratio of voltage-sensitive dyes it is necessary to reduce stray light pollution below the level of the camera's dark noise. It is thus impossible to record full-spectrum optical data sets. We address this problem by a time-multiplexed illumination, bespoke to the utilized voltage sensitive dye, to record an alternating series of intrinsic and extrinsic frames by a high-frequency CMOS sensor. These near simultaneous data series can be used to compare the mutual influence of intrinsic and extrinsic dynamics (with regards to extracorporeal functional imaging) as well as for motion compensation and thus for minimizing frame averaging, which in turn results in increased spatial precision of functional data and in a reduction of necessary experimental data sets (3R principle).

Flow Augmentation in the Myocardium by Ultrasound Cavitation of Microbubbles: Role of Shear-Mediated Purinergic Signaling

Ultrasound-mediated cavitation of microbubble contrast agents produces high intravascular shear. We hypothesized that microbubble cavitation increases myocardial microvascular perfusion through shear-dependent purinergic pathways downstream from ATP release that is immediate and sustained through cellular ATP channels such as Pannexin-1. Quantitative myocardial contrast echocardiography perfusion imaging and invivo optical imaging of ATP was performed in wild-type and Pannexin-1-deficient (Panx1-/-) mice before and 5 and 30minutes after 10minutes of ultrasound-mediated (1.3MHz, mechanical index 1.3) myocardial microbubble cavitation. Flow augmentation in a preclinical model closer to humans was evaluated in rhesus macaques undergoing myocardial contrast echocardiography perfusion imaging after high-power cavitation in the apical four-chamber plane for 10minutes. Microbubble cavitation in wild-type mice (n=7) increased myocardial perfusion by 64%±25% at 5minutes and 95%±55% at 30minutes compared with baseline (P<.05). In Panx1-/- mice (n=5), perfusion increased by 28%±26% at 5minutes (P=.04) but returned to baseline at 30minutes. Myocardial ATP signal in wild-type (n=7) mice undergoing cavitation compared with sham-treated controls (n=3) was 450-fold higher at 5minutes and 90-fold higher at 30minutes after cavitation (P<.001). The ATP signal in Panx1-/- mice (n=4) was consistently 10-fold lower than that in wild-type mice and was similar to sham controls at 30minutes. In macaques (n=8), myocardial perfusion increased twofold in the cavitation-exposed four-chamber plane, similar in degree to that produced by adenosine, but did not increase in the control two-chamber plane. Cavitation of microbubbles in the myocardial microcirculation produces an immediate release of ATP, likely from cell microporation, as well as sustained release, which is channel dependent and responsible for persistent flow augmentation. These findings provide mechanistic insight by which cavitation improves perfusion and reduces infarct size in patients with myocardial infarction.

Novel in vivo mouse model of shoulder implant infection

Animal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through invivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement. A surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 μL of 1 × 103 (e3), or 1 × 104 (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 μL sterile saline. Bacterial activity was monitored invivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed. Both inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence. Given bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII. The development of this model provides a foundation to study new therapeutics, interventions, and host modifications.

Abstract AP28: ANTITUMOR RESPONSE AFTER VACCINATION TARGETING INSULIN GROWTH FACTOR BINDING PROTEIN 2 IN A SYNGENEIC MOUSE MODEL OF OVARIAN CANCER LOCALIZED BY THREE-DIMENSIONAL IN VIVO OPTICAL IMAGING

Abstract INTRODUCTION: Ovarian cancer often disseminates as miliary implants throughout the peritoneal cavity presenting challenges for tumor quantitation in both the clinical and pre-clinical settings. The distribution of tumor implants shows affinity for areas such as the omentum and lymph nodes, which may be included in a cytoreductive surgery. Immunotherapies are being studied for ovarian cancer due to the high rate of relapse and resistance to available treatments in the recurrent setting. Pre-clinical models for ovarian cancer often do not allow meaningful assessment of novel immune-based therapies. We hypothesized vaccination targeting insulin growth factor binding protein 2 (IGFBP-2) will result in antitumor responses that can be both quantitated and localized spatially with optical imaging to identify tumor implants most resistant to vaccination. METHODS: C57BL/6/BrdCrHsd-Tyrc mice received 4 immunizations with IGFBP-2 peptides corresponding to epitopes known to favor antitumor immunity with Freund's adjuvant (treated) or with adjuvant alone (control). 5 x 106 cells of a syngeneic mouse model expressing codon-optimized firefly luciferase (luc2) known to replicate the immune microenvironment of human ovarian cancers were injected intraperitoneally. After two weeks mice were anesthetized prior to injection of D-luciferin and during imaging via inhalation of isoflurane. Mice were placed in conforming animal molds (BCAM: InVivo Analytics, Inc.) and multi-view multi-spectral image acquired with the InVivoPLOT mirror-gantry. Bioluminescent tomographic (BLt) images were reconstructed using InVivoAX (InVivo Analytics, Inc.) cloud-based software and aligned to the organ probability map (OPM), a type of statistical mouse atlas. RESULTS: Surface light intensity showed a 5.41 x 109 photons/second/mouse in control compared to 1.32 x 109 photons/second/mouse in IGFBP-2 vaccinated when measured in two dimensions. Multi-spectral tomographic reconstruction was able to show a greater than 2-fold decrease in the spatial light distribution of bioluminescent metastasis consistent with the surface light intensity distribution of the BLt image. BLt images mapped to the OPM discriminate the miliary distribution of metastases in the peritoneum. Individual metastasis in the control were shown to have a greater relative light density than the individual metastasis in the IGFBP-2 vaccinated group. CONCLUSIONS: In vivo anatomic localization of antitumor activity in a disseminated ovarian cancer mouse model can be achieved using three-dimensional optical imaging. Local differences in antitumor response to immunotherapies may be exploited to optimize their application to ovarian cancer. Identifying where tumor implants resistant to immunotherapies are sequestered may improve pre-clinical testing of immunotherapy combinations and optimally integrate them into current standard therapies. Citation Format: Denise L. Cecil, Neal Paragas, Katie M. Hitchcock-Bernhardt, Mary L. Disis, John B. Liao. ANTITUMOR RESPONSE AFTER VACCINATION TARGETING INSULIN GROWTH FACTOR BINDING PROTEIN 2 IN A SYNGENEIC MOUSE MODEL OF OVARIAN CANCER LOCALIZED BY THREE-DIMENSIONAL IN VIVO OPTICAL IMAGING [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP28.

In-vivo topical mucosal delivery of a fluorescent deoxy-glucose delineates neoplasia from normal in a preclinical model of oral epithelial neoplasia

Metabolic imaging of oral cavity mucosal surfaces could benefit early detection of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED). Fluorescent deoxy-glucose agents provide contrast for glucose metabolism similar to 18FDG-PET imaging and allow use of optical imaging, which provides high resolution and lower potential cost. However, in-vivo topical mucosal delivery of fluorescent deoxy-glucose agents without injection or tissue resection has not been shown. We introduce in-vivo optical imaging of neoplasia following mucosal delivery of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) in an OSCC/OED hamster model and demonstrate uptake into epithelium across the mucosal surface without injection or disrupting the epithelium. 2-NBDG fluorescence intensity following 30-minutes topical application was 6-fold and 4-fold higher in OSCC and OED, respectively, compared to normal mucosa. Receiver operator characteristic analysis show 83% sensitivity and 73% specificity for detection of neoplasia vs benign (normal and inflammation). Faster 2-NBDG fluorescence temporal decay in neoplasia indicated higher uptake and glucose metabolic rate than normal mucosa. Mucosal delivery of 2-NBDG by topical application to the in-vivo oral surface is feasible and delineates neoplasia from normal mucosa, providing in-vivo noninvasive molecular imaging of dysregulated glucose metabolism, which could benefit preclinical studies of carcinogenesis or be developed for use in early detection.

Odor Perception on the Two Sides of the Brain: Consistency Despite Randomness

Neurons in piriform cortex receive input from a random collection of glomeruli, resulting in odor representations that lack the stereotypic organization of the olfactory bulb. We have performed invivo optical imaging and mathematical modeling to demonstrate that correlations are retained in the transformation from bulb to piriform cortex, a feature essential for generalization across odors. Random connectivity also implies that the piriform representation of a given odor will differ among different individuals and across brain hemispheres in a single individual. We show that these different representations can nevertheless support consistent agreement about odor quality across a range of odors. Our model also demonstrates that, whereas odor discrimination and categorization require far fewer neurons than reside in piriform cortex, consistent generalization may require the full complement of piriform neurons.

In-vivo optical imaging in head and neck oncology: basic principles, clinical applications and future directions

Head and neck cancers become a severe threat to human’s health nowadays and represent the sixth most common cancer worldwide. Surgery remains the first-line choice for head and neck cancer patients. Limited resectable tissue mass and complicated anatomy structures in the head and neck region put the surgeons in a dilemma between the extensive resection and a better quality of life for the patients. Early diagnosis and treatment of the pre-malignancies, as well as real-time in vivo detection of surgical margins during en bloc resection, could be leveraged to minimize the resection of normal tissues. With the understanding of the head and neck oncology, recent advances in optical hardware and reagents have provided unique opportunities for real-time pre-malignancies and cancer imaging in the clinic or operating room. Optical imaging in the head and neck has been reported using autofluorescence imaging, targeted fluorescence imaging, high-resolution microendoscopy, narrow band imaging and the Raman spectroscopy. In this study, we reviewed the basic theories and clinical applications of optical imaging for the diagnosis and treatment in the field of head and neck oncology with the goal of identifying limitations and facilitating future advancements in the field.

Electrophoresis of polar fluorescent tracers through the nerve sheath labels neuronal populations for anatomical and functional imaging

The delivery of tracers into populations of neurons is essential to visualize their anatomy and analyze their function. In some model systems genetically-targeted expression of fluorescent proteins is the method of choice; however, these genetic tools are not available for most organisms and alternative labeling methods are very limited. Here we describe a new method for neuronal labelling by electrophoretic dye delivery from a suction electrode directly through the neuronal sheath of nerves and ganglia in insects. Polar tracer molecules were delivered into the locust auditory nerve without destroying its function, simultaneously staining peripheral sensory structures and central axonal projections. Local neuron populations could be labelled directly through the surface of the brain, and in-vivo optical imaging of sound-evoked activity was achieved through the electrophoretic delivery of calcium indicators. The method provides a new tool for studying how stimuli are processed in peripheral and central sensory pathways and is a significant advance for the study of nervous systems in non-model organisms.

Non-invasive visualization of mast cell recruitment and its effects in lung cancer by optical reporter gene imaging and glucose metabolism monitoring.

The inability to monitor the invivo dynamics of mast cells (MCs) limits the better understanding of its role in cancer progression. Here, we report on noninvasive imaging of MC migration to tumor lesions in mice and evaluation of the effects of migrated MCs on tumor progression through reporter gene-based invivo optical imaging and glucose metabolism monitoring in cancer with 18F-fluorodeoxyglucose (18F-FDG) invitro and invivo. Murine MCs (MC-9) and Lewis lung cancer cells (LLC) expressing an enhanced firefly luciferase (effluc) gene were established, termed MC-9/effluc and LLC/effluc, respectively. MC-9/effluc cell migration to LLC tumor lesions was initially detected within 1h post-transfer and distinct bioluminescence imaging signals emitted from MC-9/effluc cells were observed at tumor sites until 96h. Invivo optical imaging as well as a biodistribution study with 18F-FDG demonstrated more rapid tumor growth and upregulated glucose uptake potentially associated with MC migration to tumor lesions. These results suggest that the combination of a reporter gene-based optical imaging approach and glucose metabolism status monitoring with 18F-FDG represents a promising tool to better understand the biological role of MCs in tumor microenvironments and to develop new therapeutic drugs to regulate their involvement in enhanced tumor growth.

Evaluation of liver function and electroacupuncture efficacy of animals with alcoholic liver injury by the novel imaging methods.

Imaging methods to evaluate hepatic microcirculation (HM) and liver function (LF) by directly monitoring overall liver tissue remain lacking. This study establish imaging methods for LF that combines Laser speckle perfusion imaging (LSPI) and in vivo optical imaging (IVOI) technologies to investigate changes of hepatic microcirculation and reserve function in the animals gavaged with 50% ethanol (15 ml/kg·bw) for a model of acute alcoholic liver injury (ALI), and for evaluation of electroacupuncture (EA) effect. The liver blood perfusion and indocyanine green (ICG) distribution were observe by LSPI and IVOI separately. After EA, the livers were collected to measure the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), thromboxane A (TXA2), prostacyclin (PGI2) and endothelin (ET). The acquisitions of newly established LSPI of liver and ICG in vivo fluorescence imaging (ICG-IVFI), combining the results of other indexes showed: hepatic microcirculation perfusion (HMP) significantly reduced, ICG metabolism reduced, and ALT/AST increased in animal model with acute ALI. EA can reverse these changes. The use of LSPI of liver and ICG-IVFI, which was novel imaging methods for LF established in this study, could display the LF characteristics of ALI and the EA efficacy.

Persistent luminescence of Eu, Mn, Dy doped calcium phosphates for in-vivo optical imaging

Biocompatible nanoparticles possessing persistent luminescence properties offer attractive possibilities for in vivo imaging applications as it allows an excitation of the sensors outside the animal before injection and a long-lasting emission of light. Here we report the development of highly biocompatible calcium phosphate nanoparticles doped with europium, Mn2+ and Ln3+ (Ln3+=Dy3+, Pr3+) ions synthesized by hydrothermal route and tailored to present red-near infrared persistent luminescence after UV excitation. Nanosize biphasic HAp/β-TCP compounds with sphere and rod-shaped were obtained. Two emission bands in the red-near infrared range were observed and attributed to 4T1→6A1 transitions of Mn2+ ions in HAp/β-TCP. An annealing treatment in reductive atmosphere post-synthesis was essential to reveal persistent luminescence properties. Indeed, such thermal treatment allows reducing Eu3+ ions in Eu2+ ions and generating required defaults as oxygen vacancies in the crystal necessary for red emission in accordance with persistent luminescence mechanism. These nanoparticles have been tested for the first time for in vivo imaging on small animal as proof of concept of prospective highly biocompatible nanoprobes.

Dissecting the Role of Smooth Muscle Cells versus Pericytes in Regulating Cerebral Blood Flow Using In Vivo Optical Imaging

The brain regulates blood flow to match energy demand to nutrient supply. In this issue of Neuron, using invivo optical imaging and optogenetics, Hill etal. (2015) report that arteriolar smooth muscle cells are key players in regulating cerebral blood flow in the healthy state and contribute to the "no reflow" phenomenon after ischemic stroke.

In vivo imaging of MMP-13 activity in the murine destabilised medial meniscus surgical model of osteoarthritis

To detect and determine disease severity of osteoarthritis (OA) using a probe activated by matrix metalloproteinase-13 (MMP-13) invivo in the murine destabilised medial meniscus (DMM) surgical model of OA. We have previously described MMP12ap and MMP13ap, internally quenched fluorescent peptide substrate probes that are activated respectively by MMP-12 and MMP-13. Here we used these probes to follow enzyme activity invivo in mice knees 4, 6 and 8 weeks following DMM surgery. After invivo optical imaging, disease severity was determined through traditional histological analysis. The amount of probe activation was analysed for discrimination between DMM, contralateral and sham operated knees, as well as for congruence between activity and histological damage. There was no specific activation of MMP12ap at the time points observed between sham operated and DMM operated, or their respective contralateral joints. The activation of the MMP13ap in the DMM model was highest 6 weeks after surgery, but was only specific compared against sham surgery 8 weeks after surgery (1.5-fold increase). The activation of MMP13ap correlated with histological damage 6 and 8 weeks after surgery, with correlations of 0.484 (P=0.0032) and 0.478 respectively (P=0.0049). This correlation dropped to 0.218 (P=0.011) if all data were considered. The current MMP-13 activity probe is suitable for the discrimination between DMM and sham or contralateral knees 8 weeks after surgery, when cartilage loss is typified by the appearance of small fissures up to the tidemark, but not earlier. This activity correlates with the histological damage observed.

Abstract 5688: Multifunctional gold nanoparticles linked with aptamers and fluorophores for breast cancer imaging and therapy

Abstract Breast cancer is a leading cause of mortality among women worldwide and there is a need for improved methods for early detection, new therapies that are more effective with fewer side effects, and personalized treatment options. Nucleic acid aptamers are synthetic oligonucleotides that bind to specific target proteins and have potential for both imaging and therapy. They have a targeting mechanism similar to monoclonal antibodies (i.e. shape-specific recognition), but may have substantial advantages, including easier synthesis and storage, better tumor penetration, and non-immunogenicity. Previously, we developed AS1411 (now renamed ACT-GRO-777), a nucleolin-binding DNA aptamer that has antiproliferative activity against cancer cells with little effect on non-malignant cells. AS1411 was the first anticancer aptamer to be tested in human clinical trials, which indicated promising activity with no evidence of serious side effects. The molecular target for AS1411 is nucleolin, a protein highly expressed on the surface of breast cancer cells and breast tumor-associated endothelial cells, suggesting the potential utility of AS1411 in this disease. Here we report on new research in which we conjugated gold nanoparticles (GNPs) to AS1411 and assessed their potential for breast cancer imaging and therapy. The antiproliferative activity of AS1411 linked to 5 nm gold nanoparticles (GNP) was determined by MTT and clonogenic assays. The GI50 value for AS1411-GNPs against breast cancer cell lines was less than 100 nM (aptamer concentration), which is at least 20-fold lower than unconjugated AS1411. Moreover, AS1411-GNPs retained the cancer-selectivity of AS1411 and had no effect on non-malignant breast cells. Confocal microscopy revealed increased uptake in breast cancer cells for GNP-AS1411 compared to GNP alone or GNP conjugated to a control oligonucleotide. In addition, GNP-AS1411 potently induced breast cancer cell vacuolization and death, similar to that seen at higher concentrations of AS1411. Furthermore, in vivo studies in nude mice with established MDA-MB-231 xenografts have shown that systemic administration of AS1411-GNPs for 12 days could completely inhibit tumor growth in a specific manner. We further examined if the GNP-AS1411 conjugated to fluorophore Cy5 has a potential for in-vivo optical imaging to specifically detect the tumor in established breast cancer xenograft model. Preliminary data from biodistribution studies suggest that GNP-AS1411 can very efficiently detect tumors using in vivo imaging modalities. Altogether, our data suggest that this strategy could be used to develop multifunctional tumor-targeting nanoparticles that can serve as imaging agents to detect breast cancers or monitor clinical response, as well as to specifically deliver therapeutic agents to the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5688. doi:1538-7445.AM2012-5688