Ines Gemeinhardt

Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation.

We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ~70 sulfate groups) targets factors of inflammation (IC(50) of 3-6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1-3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

Extra Domain B Fibronectin as a Target for Near-Infrared Fluorescence Imaging of Rheumatoid Arthritis Affected Joints In Vivo

We investigated a molecular imaging approach for the detection of collagen-induced arthritis in rats by targeting the extra domain B (ED-B) of the extracellular matrix protein fibronectin. ED-B is a highly conserved domain (identical in human and rats) that is produced by alternative splicing during embryonic development and during vascular remodeling such as angiogenesis. The hallmark of rheumatoid arthritis is synovitis leading to both angiogenesis in the synovium and the promotion of cartilage and bone disruption. For in vivo diagnostics, the ED-B-binding single-chain antibody fragment AP39 was used as a targeting probe. It was covalently linked to the near-infrared dye tetrasulfocyanine (TSC) to be visualized by near-infrared fluorescence imaging. The resulting AP39-TSC conjugate was intravenously administered to rats with collagen-induced arthritis and the respective controls. Ovalbumin-TSC was used as control conjugate. Optical imaging over a time period of 24 hours using a planar imaging setup resulted in a clear enhancement of fluorescence intensity in joints with moderate to severe arthritis compared with control joints between 3 and 8 hours postinjection. Given that AP39 is a fully human antibody fragment, this molecular imaging approach for arthritis detection might be translated to humans.

Cellular Uptake of Magnetic Nanoparticles Quantified by Magnetic Particle Spectroscopy

The quantification of magnetic iron oxide nanoparticles in biological systems (cells, tissues and organs) is of vital importance in the development of novel biomedical applications such as magnetofection, drug targeting or hyperthermia. Among several techniques established to detect iron in tissue, the recently developed technique of magnetic particle spectroscopy (MPS) provides signals that are specific for magnetic nanoparticles. MPS utilizes the non-linear response of an MNP sample to a strong sinusoidal excitation field of up to 25 mT amplitude and 25 kHz. We demonstrate the feasibility of this technique to quantify nanoparticle uptake in cells using a commercial magnetic particle spectrometer (Bruker BioSpin).

Near-infrared fluorescence imaging of experimentally collagen-induced arthritis in rats using the nonspecific dye tetrasulfocyanine in comparison with gadolinium-based contrast-enhanced magnetic resonance imaging, histology, and clinical score.

Abstract. Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p<0.005) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, INF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81%) than for MRI (100%/91%). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.

In vivo therapy monitoring of experimental rheumatoid arthritis in rats using near-infrared fluorescence imaging.

Abstract. An in vivo near-infrared fluorescence (NIRF) imaging technique is described for therapy monitoring of ankle joints affected by collagen-induced arthritis, a model of human rheumatoid arthritis. Arthritis was induced in rats by intradermal injections of collagen and Freund’s incomplete adjuvant. For in vivo imaging, the nonspecific NIR dye tetrasulfocyanine (TSC) was used. Prior to and after treatment with a nonsteroidal anti-inflammatory drug, meloxicam, or analgesic drug, tramadol hydrochloride (which served as no-therapy control), normalized fluorescence intensities of each ankle joint were measured. Additionally, each ankle joint was characterized by clinical arthritis scoring and histopathology. Over a 3-week treatment period, a significant difference in disease progression between animals treated with meloxicam and tramadol hydrochloride was detected. A statistically significant improvement in ankle joint pathology from high- or moderate-grade to moderate- or low-grade upon meloxicam therapy, as determined by clinical evaluation, translated into a significant decrease in fluorescence intensity. In contrast, all arthritic joints of the no-therapy control group deteriorated to high-grade arthritis with high-fluorescence intensities in NIRF imaging.

Quantification of Magnetic Nanoparticle Uptake in Cells by Temperature Dependent Magnetorelaxometry

Typically, magnetic iron oxide nanoparticles (MNP) with core diameter in the range of about 16 nm to 22 nm are accessible by magnetorelaxometric (MRX) measurements at room temperature whereas the relaxation of smaller particles is too fast to be observed with a conventional MRX setup. In order to extend the size limitation towards smaller particles, we suggest applying temperature dependent magnetorelaxometry (TMRX). In this study, we outline and validate the procedure experimentally for temperatures between 5 K and 200 K on in-vitro preparations of MNP using a conventional MPMS SQUID magnetometer. On this basis, we applied TMRX for the in-vitro quantification of small sized MNP uptake by tumor cells, i.e. on HeLa and Jurkat tumor cell lines, reaching a detection limit of about 100 ng. We further showed that TMRX signals are characteristic for particular MNP preparations, opening the possibility to observe changes in the particle size distribution during the uptake of MNP by a biological system.

Influence of the blood exposure time in dynamic hemocompatibility testing on coagulation and C5a activation

Within the hemocompatibility testing portfolio of medical devices a range of dynamic models were established in recent years. In contrast to the static hemocompatibility testing method the dynamic models allow considering the impact of hemorheological and hemodynamic blood characteristics on the hemocompatibility of medical devices. Unfortunately the EN DIN ISO 10993-4 for the biological evaluation of medical devices for interaction with blood gives no hints towards the period of time during which the medical devices should be exposed to the blood in these tests. To examine whether different exposure times impact the comparability of hemocompatibility test results low density polyethylene (LD-PE) tubes and nitinol stents were tested exemplarily in a closed loop model for changes of the fibrinogen content, the prothrombin time, the thrombin time, and the C5a activity after 30 and 90 min exposure to the blood. Low density polyethylene was used as negative control because it is one of the European reference materials for hemocompatibility testing. After 90 min blood exposure to the LD-PE tubing and the nitinol stents the prothrombin time was significantly longer and the fibrinogen content significantly lower (p < 0.05) than after 30 min. In contrast the thrombin time and the C5a were comparable after 30 and 90 min blood exposure time. These results might recommend to an initial 30 min exposure time, which is followed by a 90 min exposure time in cases of unclear results.

SAT0073 Theranostic approach in arthritis: A feasibility study in rats

Background Assessment of arthritis using fluorescence optical imaging (FOI) in combination with the fluorophore indocyanine green (ICG) is now clinically established [1]. Beyond imaging of inflammation, FOI also provides the opportunity to monitor drug targeting of biological therapeutics into inflamed joints. Quantitative information of drug targeting would allow for patient selection and individual treatment schedules. Objectives We report the first synthesis of etanercept-ICG-conjugates and the quantitative monitoring of drug targeting into inflamed joints of collagen II induced arthritis using a modified fluorescent camera system (Xiralite). Methods Inflammatory arthritis was induced by twice injections of bovine collagen II (day 0, day 7) in female Lewis rats. Rats were monitored for joint swelling, pain, redness and mobility (score 0 to 3). The TNFa binding protein etanercept (Enbrel®, Pfizer) was chemically conjugated with an ICG derivative suited for labeling of proteins. The etanercept-6S-ICG (Eta-6S-ICG) conjugate was photophysically characterized and tested for binding affinity The fluorescence camera system Xiralite (mivenion GmbH, Germany) [1] was modified to enable imaging of rats. On day 14 after first injection of collagen, Eta-6S-ICG (n=6), IgG-6S-ICG (n=3), or the inflammation-targeted macromolecule polyglycerolsulfate-6S-ICG (dPGS-6S-ICG, n=5) [2] were s.c. or i.v. injected in a dose of 10 nMol/kg b.w. Fluorescence measurement was performed before and 1 h, 3 h, 24 h and 48 h after injection. Fluorescence intensity (FI) was measured in ROI over the tibio-tarsal and interphalangeal articulations and compared with healthy reference ROI. In addition, targeting was studied in rat tissues 3 or 24 h post injection of Eta-6S-, IgG-6S- or dPGS-6S-indocarbocyanine conjugates using fluorescence histology. Results Binding affinity of etanercept was not attenuated by chemical conjugation with 6S-ICG. Eta-6S-ICG exerts high fluorescence quantum yield compared with ICG. FI was detected already 24 hours after administration of Eta-6S-ICG and further increased until 48 hours after injection. Significant FI at 48 hours after injection of Eta-6S-ICG was measured in tibio-tarsal joints (score 1: 15.8±2.9; score 3: 31.0±4.6) while FI in interphalangeal joints was low (score 1: 6.3±1.0; score 3: 7.7±1.2). In contrast, dPGS-6S-ICG exhibited high FI in both tibio-tarsal joints (score 1: 23.5±4.9; score 3: 69.8±17.5) and interphalangeal joints (score 1: 20.9±5.8; score 3: 37.0±4.7). Both Eta-6S- ICG and dPGS-6S-ICG show strong targeting to inflamed synovial tissue and cartilage compared with IgG-6S-ICG. Conclusions FOI and labeling of macromolecular therapeutics with novel ICG derivatives provide a novel rationale for sensitive and quantitative monitoring of arthritis therapy. Further studies have to determine the effect of varying molecular size on the degree of targeting of inflamed joints. References Werner S et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis doi:10.1136/ard.2010.148288. Licha K et al. Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation. Bioconjug Chem; doi:10.1021/bc2002727 Disclosure of Interest P. Welker Employee of: mivenion GmbH, K. Licha Shareholder of: mivenion GmbH, I. Gemeinhardt: None Declared, M. Bahner Shareholder of: mivenion GmbH, J. Berger Employee of: mivenion GmbH, D. Mangoldt Employee of: mivenion GmbH, J. Schnorr: None Declared, M. Schirner Shareholder of: mivenion GmbH

Fluorescence imaging of experimental rheumatoid arthritis in vivo using a fast flying-spot scanner

We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.