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Gunter Almer,1,* Kelli L Summers,1,2,* Bernhard Scheicher,2 Josef Kellner,3 Ingeborg Stelzer,1 Gerd Leitinger,4,5 Anna Gries,3 Ruth Prassl,6 Andreas Zimmer,2 Harald Mangge1,7 1Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; 2Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz, Graz, Austria; 3Institute of Physiological Chemistry, Medical University of Graz, Graz, Austria; 4Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria; 5Center for Medical Research (ZMF), Medical University of Graz, Graz, Austria; 6Institute of Biophysics, Medical University of Graz, Graz, Austria; 7BioTechMed, Graz, Austria*These authors contributed equally to the study Abstract: Atherosclerosis (AS) is one of the leading causes of mortality in high-income countries. Early diagnosis of vulnerable atherosclerotic lesions is one of the biggest challenges currently facing cardiovascular medicine. The present study focuses on developing targeted nanoparticles (NPs) in order to improve the detection of vulnerable atherosclerotic-plaques. Various biomarkers involved in the pathogenesis of atherosclerotic-plaques have been identified and one of these promising candidates for diagnostic targeting is interleukin 10 (IL10). IL10 has been shown to be a key anti-inflammatory responding cytokine in the early stages of atherogenesis, and has already been used for therapeutic interventions in humans and mice. IL10, the targeting sequence, was coupled to two different types of NPs: protamine-oligonucleotide NPs (proticles) and sterically stabilized liposomes in order to address the question of whether the recognition and detection of atherosclerotic-lesions is primarily determined by the targeting sequence itself, or whether it depends on the NP carrier system to which the biomarker is coupled. Each IL10-targeted N

Gunter Almer,1,* Kelli L Summers,1,2,* Bernhard Scheicher,2 Josef Kellner,3 Ingeborg Stelzer,1 Gerd Leitinger,4,5 Anna Gries,3 Ruth Prassl,6 Andreas Zimmer,2 Harald Mangge1,7 1Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; 2Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz, Graz, Austria; 3Institute of Physiological Chemistry, Medical University of Graz, Graz, Austria; 4Research Unit Electron Microscopic Techniques, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria; 5Center for Medical Research (ZMF), Medical University of Graz, Graz, Austria; 6Institute of Biophysics, Medical University of Graz, Graz, Austria; 7BioTechMed, Graz, Austria*These authors contributed equally to the study Abstract: Atherosclerosis (AS) is one of the leading causes of mortality in high-income countries. Early diagnosis of vulnerable atherosclerotic lesions is one of the biggest challenges currently facing cardiovascular medicine. The present study focuses on developing targeted nanoparticles (NPs) in order to improve the detection of vulnerable atherosclerotic-plaques. Various biomarkers involved in the pathogenesis of atherosclerotic-plaques have been identified and one of these promising candidates for diagnostic targeting is interleukin 10 (IL10). IL10 has been shown to be a key anti-inflammatory responding cytokine in the early stages of atherogenesis, and has already been used for therapeutic interventions in humans and mice. IL10, the targeting sequence, was coupled to two different types of NPs: protamine-oligonucleotide NPs (proticles) and sterically stabilized liposomes in order to address the question of whether the recognition and detection of atherosclerotic-lesions is primarily determined by the targeting sequence itself, or whether it depends on the NP carrier system to which the biomarker is coupled. Each IL10-targeted N

Daniela Frascione,1 Clemens Diwoky,2 Gunter Almer,1,3 Peter Opriessnig,2 Caroline Vonach,1 Kerstin Gradauer,1 Gerd Leitinger,4 Harald Mangge,3 Rudolf Stollberger,2 Ruth Prassl1,51Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria; 2Institute of Medical Engineering, University of Technology, Graz, Austria; 3Clinical Institute for Medical and Chemical Laboratory Diagnosis (CIMCL), Medical University, Graz, Austria; 4Institute of Cell Biology, Histology and Embryology, Medical University, Graz, Austria; 5Ludwig Boltzmann Institute for Lung Vascular Research, Graz, AustriaBackground: Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.Methods: Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the sign

Daniela Frascione,1 Clemens Diwoky,2 Gunter Almer,1,3 Peter Opriessnig,2 Caroline Vonach,1 Kerstin Gradauer,1 Gerd Leitinger,4 Harald Mangge,3 Rudolf Stollberger,2 Ruth Prassl1,51Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz, Austria; 2Institute of Medical Engineering, University of Technology, Graz, Austria; 3Clinical Institute for Medical and Chemical Laboratory Diagnosis (CIMCL), Medical University, Graz, Austria; 4Institute of Cell Biology, Histology and Embryology, Medical University, Graz, Austria; 5Ludwig Boltzmann Institute for Lung Vascular Research, Graz, AustriaBackground: Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magnetic and/or paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI). MLs have an advantage over free magnetic nanocores, in that various functional groups can be attached to the surface of liposomes for ligand-specific targeting. We have synthesized PEG-coated sterically-stabilized magnetic liposomes (sMLs) containing ultrasmall superparamagnetic iron oxides (USPIOs) with the aim of generating stable liposomal carriers equipped with a high payload of USPIOs for enhanced MRI contrast.Methods: Regarding iron oxide nanoparticles, we have applied two different commercially available surface-coated USPIOs; sMLs synthesized and loaded with USPIOs were compared in terms of magnetization and colloidal stability. The average diameter size, morphology, phospholipid membrane fluidity, and the iron content of the sMLs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence polarization, and absorption spectroscopy, respectively. A colorimetric assay using potassium thiocyanate (KSCN) was performed to evaluate the encapsulation efficiency (EE%) to express the amount of iron enclosed into a liposome. Subsequently, MRI measurements were carried out in vitro in agarose gel phantoms to evaluate the sign

Gunter Almer1,6, Karin Wernig2, Matthias Saba-Lepek3, Samih Haj-Yahya1, Johannes Rattenberger4, Julian Wagner4, Kerstin Gradauer3, Daniela Frascione3, Georg Pabst3, Gerd Leitinger5, Harald Mangge1, Andreas Zimmer2, Ruth Prassl31Clinical Institute of Medical and Chemical Laboratory Diagnostics, 2Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz, 3Institute of Biophysics and Nanosystems Research, Austrian Academy of Science, 4Institute for Electron Microscopy and Fine Structure Research, Graz University of Technology, 5Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 6Center for Medical Research, Medical University of Graz, AustriaBackground: Atherosclerosis is a leading cause of mortality in the Western world, and plaque diagnosis is still a challenge in cardiovascular medicine. The main focus of this study was to make atherosclerotic plaques visible using targeted nanoparticles for improved imaging. Today various biomarkers are known to be involved in the pathophysiologic scenario of atherosclerotic plaques. One promising new candidate is the globular domain of the adipocytokine adiponectin (gAd), which was used as a targeting sequence in this study.Methods: gAd was coupled to two different types of nanoparticles, namely protamine-oligonucleotide nanoparticles, known as proticles, and sterically stabilized liposomes. Both gAd-targeted nanoparticles were investigated for their potency to characterize critical scenarios within early and advanced atherosclerotic plaque lesions using an atherosclerotic mouse model. Aortic tissue from wild type and apolipoprotein E-deficient mice, both fed a high-fat diet, were stained with either fluorescent-labeled gAd or gAd-coupled nanoparticles. Ex vivo imaging was performed using confocal laser scanning microscopy.Results: gAd-targeted sterically stabilized liposomes generated a strong signal by accumulating at the surface of atherosclerotic plaques, wh

Gunter Almer1,6, Karin Wernig2, Matthias Saba-Lepek3, Samih Haj-Yahya1, Johannes Rattenberger4, Julian Wagner4, Kerstin Gradauer3, Daniela Frascione3, Georg Pabst3, Gerd Leitinger5, Harald Mangge1, Andreas Zimmer2, Ruth Prassl31Clinical Institute of Medical and Chemical Laboratory Diagnostics, 2Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz, 3Institute of Biophysics and Nanosystems Research, Austrian Academy of Science, 4Institute for Electron Microscopy and Fine Structure Research, Graz University of Technology, 5Institute of Cell Biology, Histology and Embryology, Medical University of Graz, 6Center for Medical Research, Medical University of Graz, AustriaBackground: Atherosclerosis is a leading cause of mortality in the Western world, and plaque diagnosis is still a challenge in cardiovascular medicine. The main focus of this study was to make atherosclerotic plaques visible using targeted nanoparticles for improved imaging. Today various biomarkers are known to be involved in the pathophysiologic scenario of atherosclerotic plaques. One promising new candidate is the globular domain of the adipocytokine adiponectin (gAd), which was used as a targeting sequence in this study.Methods: gAd was coupled to two different types of nanoparticles, namely protamine-oligonucleotide nanoparticles, known as proticles, and sterically stabilized liposomes. Both gAd-targeted nanoparticles were investigated for their potency to characterize critical scenarios within early and advanced atherosclerotic plaque lesions using an atherosclerotic mouse model. Aortic tissue from wild type and apolipoprotein E-deficient mice, both fed a high-fat diet, were stained with either fluorescent-labeled gAd or gAd-coupled nanoparticles. Ex vivo imaging was performed using confocal laser scanning microscopy.Results: gAd-targeted sterically stabilized liposomes generated a strong signal by accumulating at the surface of atherosclerotic plaques, wh

Gunter Almer1, Matthias Saba-Lepek2, Samih Haj-Yahya1, Eva Rohde3, Dirk Strunk4, Eleonore Fröhlich5, Ruth Prassl2, Harald Mangge11Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, 2Institute of Biophysics and Nanosystems Research, Austrian Academy of Science, 3University Clinic of Blood Group Serology and Transfusion Medicine, 4University Clinic of Hematology, 5Center for Medical Research, Medical University of Graz, Graz, AustriaBackground: Adiponectin, an adipocyte-specific plasma protein, has been shown to accumulate in injured endothelial cells during development of atherosclerotic lesions. In this study, we investigated the potential of different adiponectin subfractions with special emphasis on globular adiponectin (gAd) to recognize and visualize atherosclerotic lesions.Methods: Recombinant mouse gAd and subfractions of full-length adiponectin (ie, trimeric, hexameric, and oligomeric forms) were fluorescence-labeled. Aortas of wild-type and apoprotein E-deficient mice fed a high cholesterol diet were dissected and incubated with the labeled biomarkers. Imaging was performed using confocal laser scanning microscopy.Results: Confocal laser scanning microscopic images showed that gAd binds more strongly to atherosclerotic plaques than full-length adiponectin subfractions. Further, we showed that gAd accumulates preferentially in endothelial cells and the fibrous cap area of plaques. Here we demonstrate for the first time that gAd recognizes atherosclerotic plaques on aortic sections of apoprotein E-deficient mice.Conclusion: These results suggest that gAd, in addition to its physiological properties, is also suitable as a target molecule for prospective diagnostic strategies in imaging atherosclerotic lesions.Keywords: adiponectin subfractions, atherosclerosis, fibrous cap, globular adiponectin, vascular imaging