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Alexander Sekita,1 Harald Unterweger,1 Sonja Berg,1 Sabine Ohlmeyer,2 Tobias Bäuerle,3 Kang H Zheng,4 Bram F Coolen,5 Aart J Nederveen,6 Claudia Cabella,7 Silvia Rossi,7 Erik SG Stroes,4 Christoph Alexiou,1 Stefan Lyer,1,* Iwona Cicha1,* 1ENT-Department, Section of Experimental Oncology Und Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany; 2Institute of Radiology, Universitätsklinikum Erlangen, Erlangen, Germany; 3Preclinical Imaging Platform Erlangen (PIPE), Universitätsklinikum Erlangen, Erlangen, Germany; 4Department of Vascular Medicine, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 5Department of Biomedical Engineering and Physics, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 6Department of Radiology and Nuclear Medicine, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 7Bracco Imaging SpA, Centro Ricerche Bracco, Colleretto Giacosa, Turin, Italy*These authors contributed equally to this workCorrespondence: Iwona Cicha, Universitätsklinikum Erlangen, Glückstr, 10a, Erlangen, 91054, Germany, Tel +49 9131 85 43953, Fax +49 9131 85 34828, Email Iwona.Cicha@uk-erlangen.dePurpose: In this study, a detailed characterization of a rabbit model of atherosclerosis was performed to assess the optimal time frame for evaluating plaque vulnerability using superparamagnetic iron oxide nanoparticle (SPION)-enhanced magnetic resonance imaging (MRI).Methods: The progression of atherosclerosis induced by ballooning and a high-cholesterol diet was monitored using angiography, and the resulting plaques were characterized using immunohistochemistry and histology. Morphometric analyses were performed to evaluate plaque size and vulnerability features. The accumulation of SPIONs (novel dextran-coated SPIONDex and ferumoxytol) in atherosclerotic plaques was investigated by histology and MRI and correlated with plaque age and vulnerability. Toxicity of SPIONDex was evaluated in rats.Results: Weak positive correlations were detected between plaque age and intima thickness, and total macrophage load. A strong negative correlation was observed between the minimum fibrous cap thickness and plaque age as well as the mean macrophage load. The accumulation of SPION in the atherosclerotic plaques was detected by MRI 24 h after administration and was subsequently confirmed by Prussian blue staining of histological specimens. Positive correlations between Prussian blue signal in atherosclerotic plaques, plaque age, and macrophage load were detected. Very little iron was observed in the histological sections of the heart and kidney, whereas strong staining of SPIONDex and ferumoxytol was detected in the spleen and liver. In contrast to ferumoxytol, SPIONDex administration in rabbits was well tolerated without inducing hypersensitivity. The maximum tolerated dose in rat model was higher than 100 mg Fe/kg.Conclusion: Older atherosclerotic plaques with vulnerable features in rabbits are a useful tool for investigating iron oxide-based contrast agents for MRI. Based on the experimental data, SPIONDex particles constitute a promising candidate for further clinical translation as a safe formulation that offers the possibility of repeated administration free from the risks associated with other types of magnetic contrast agents.Keywords: atherosclerosis, plaque vulnerability, rabbit model, macrophages, histology, tissue iron, SPION-enhanced MRI

Harald Unterweger,1,* Christina Janko,1,* Tamara Folk,1 Iwona Cicha,1 Noémi Kovács,2 Gyula Gyebnár,3 Ildikó Horváth,4 Domokos Máthé,2,3 Kang H Zheng,5 Bram F Coolen,6 Erik Stroes,5 János Szebeni,7,8 Christoph Alexiou,1 László Dézsi,7,8,* Stefan Lyer1,* 1ENT-Department, Section of Experimental Oncology und Nanomedicine (SEON), Universitätsklinikum Erlangen, Erlangen, Germany; 2Hungarian Centre of Excellence for Molecular Medicine, Semmelweis University, Budapest, Hungary; 3Medical Imaging Centre, Semmelweis University, Budapest, Hungary; 4Department Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary; 5Department of Vascular Medicine, Amsterdam University Medical Centers, Amsterdam Cardiovascular Sciences, University of Amsterdam, Amsterdam, the Netherlands; 6Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam Cardiovascular Sciences, University of Amsterdam, Amsterdam, the Netherlands; 7Nanomedicine Research and Education Center, Institute of Translational Medicine, Semmelweis University, Budapest, Hungary; 8SeroScience Ltd, Budapest, Hungary*These authors contributed equally to this workCorrespondence: Harald Unterweger, Universitätsklinikum Erlangen, Glueckstr. 10a, Erlangen, 91054, Germany, Tel +49 9131 85-33142, Fax +49 9131 85-34828, Email harald.unterweger@uk-erlangen.deIntroduction: One of the major challenges in the clinical translation of nanoparticles is the development of formulations combining favorable efficacy and optimal safety. In the past, iron oxide nanoparticles have been introduced as an alternative for gadolinium-containing contrast agents; however, candidates available at the time were not free from adverse effects.Methods: Following the development of a potent iron oxide-based contrast agent SPIONDex, we now performed a systematic comparison of this formulation with the conventional contrast agent ferucarbotran and with ferumoxytol, taking into consideration their physicochemical characteristics, bio- and hemocompatibility in vitro and in vivo, as well as their liver imaging properties in rats.Results: The results demonstrated superior in vitro cyto-, hemo- and immunocompatibility of SPIONDex in comparison to the other two formulations. Intravenous administration of ferucarbotran or ferumoxytol induced strong complement activation-related pseudoallergy in pigs. In contrast, SPIONDex did not elicit any hypersensitivity reactions in the experimental animals. In a rat model, comparable liver imaging properties, but a faster clearance was demonstrated for SPIONDex.Conclusion: The results indicate that SPIONDex possess an exceptional safety compared to the other two formulations, making them a promising candidate for further clinical translation.Keywords: magnetic resonance imaging, MRI, nanomedicine, nanoparticles, complement activation, CARPA

Ralf P Friedrich,1 Mona Kappes,1 Iwona Cicha,1 Rainer Tietze,1 Christian Braun,2 Regine Schneider-Stock,3 Roland Nagy,4 Christoph Alexiou,1 Christina Janko1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, Erlangen, 91054, Germany; 2Institute of Legal Medicine, Ludwig-Maximilians-Universität München, München, 80336, Germany; 3Experimental Tumor Pathology, Institute of Pathology, University Hospital, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91054, Germany; 4Department Elektrotechnik-Elektronik-Informationstechnik (EEI), Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, 91058, GermanyCorrespondence: Christina Janko, Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, Glückstrasse 10a, Erlangen, 91054, Germany, Tel +49 9131 85 33142, Fax +49 9131 85 34808, Email christina.janko@uk-erlangen.deAbstract: Label-free detection of nanoparticles is essential for a thorough evaluation of their cellular effects. In particular, nanoparticles intended for medical applications must be carefully analyzed in terms of their interactions with cells, tissues, and organs. Since the labeling causes a strong change in the physicochemical properties and thus also alters the interactions of the particles with the surrounding tissue, the use of fluorescently labeled particles is inadequate to characterize the effects of unlabeled particles. Further, labeling may affect cellular uptake and biocompatibility of nanoparticles. Thus, label-free techniques have been recently developed and implemented to ensure a reliable characterization of nanoparticles.This review provides an overview of frequently used label-free visualization techniques and highlights recent studies on the development and usage of microscopy systems based on reflectance, darkfield, differential interference contrast, optical coherence, photothermal, holographic, photoacoustic, total internal reflection, surface plasmon resonance, Rayleigh light scattering, hyperspectral and reflectance structured illumination imaging. Using these imaging modalities, there is a strong enhancement in the reliability of experiments concerning cellular uptake and biocompatibility of nanoparticles, which is crucial for preclinical evaluations and future medical applications.Keywords: label free imaging, nanoparticle detection, non-fluorescent imaging, reflectance imaging, holotomography, scattering microscopy

Jasmin Matuszak, Barbara Lutz, Aleksander Sekita, Jan Zaloga, Christoph Alexiou, Stefan Lyer,* Iwona Cicha* Cardiovascular Nanomedicine Unit, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-endowed Professorship for Nanomedicine, ENT Department, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany *These authors contributed equally to this work Introduction: Magnetic drug targeting utilizes superparamagnetic iron oxide nanoparticles (SPIONs) to accumulate drugs in specified vasculature regions.Methods: We produced SPIONs conjugated with dexamethasone phosphate (SPION-DEXA). The efficacy of magnetic drug targeting was investigated in a rabbit model of atherosclerosis induced by balloon injury and high cholesterol diet.Results: In vitro, SPION-DEXA were well-tolerated by endothelial cells. SPION-DEXA were internalized by human peripheral blood mononuclear cells and induced CD163 expression comparable with the free drug. In vivo, magnetic targeting of SPIONs to abdominal aorta was confirmed by histology. Upon vascular injury followed by high-cholesterol diet, early administration of SPION-DEXA enhanced the inflammatory burden in the plaques. Increased macrophage content and larger intima–media thickness were observed in animals treated with SPION-DEXA compared with controls. In advanced atherosclerosis, no beneficial effect of local glucocorticoid therapy was detectable.Conclusion: Magnetic drug targeting represents an efficient platform to deliver drugs to diseased arteries in vivo. However, targeting of vascular injury in the lipid-rich environment using dexamethasone-conjugated SPIONs may cause accelerated inflammatory response. Keywords: magnetic nanoparticles, magnetic drug targeting, rabbit model of atherosclerosis, dexamethasone, macrophage accumulation

Harald Unterweger,1 László Dézsi,2,3 Jasmin Matuszak,1 Christina Janko,1 Marina Poettler,1 Jutta Jordan,4 Tobias Bäuerle,4 János Szebeni,2,3 Tobias Fey,5 Aldo R Boccaccini,6 Christoph Alexiou,1 Iwona Cicha1 1ENT Department, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, Erlangen, Germany; 2Nanomedicine Research and Education Center, Institute of Pathophysiology, Semmelweis University, Budapest, Hungary; 3SeroScience Ltd., Budapest, Hungary; 4Institute of Radiology, Preclinical Imaging Platform Erlangen, Universitätsklinikum Erlangen, Erlangen, Germany; 5Institute of Glass and Ceramics, Department of Materials Science and Engineering, University Erlangen-Nuremberg, Erlangen, Germany; 6Institute of Biomaterials, Department of Materials Science and Engineering, University Erlangen-Nuremberg, Erlangen, Germany Background: Rising criticism of currently available contrast agents for magnetic resonance imaging, either due to their side effects or limited possibilities in terms of functional imaging, evoked the need for safer and more versatile agents. We previously demonstrated the suitability of novel dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) for biomedical applications in terms of safety and biocompatibility. Methods: In the present study, we investigated the size-dependent cross-linking process of these particles as well as the size dependency of their imaging properties. For the latter purpose, we adopted a simple and easy-to-perform experiment to estimate the relaxivity of the particles. Furthermore, we performed an extensive analysis of the particles’ storage stability under different temperature conditions, showing their superb stability and the lack of any signs of agglomeration or sedimentation during a 12 week period. Results: Independent of their size, SPIONDex displayed no irritation potential in a chick chorioallantoic membrane assay. Cell uptake studies of ultra-small (30 nm) SPIONDex confirmed their internalization by macrophages, but not by non-phagocytic cells. Additionally, complement activation-related pseudoallergy (CARPA) experiments in pigs treated with ultra-small SPIONDex indicated the absence of hypersensitivity reactions. Conclusion: These results emphasize the exceptional safety of SPIONDex, setting them apart from the existing SPION-based contrast agents and making them a very promising candidate for further clinical development. Keywords: CARPA, cross-linking, irritation potential, MRI, SPION, storage stability

Harald Unterweger,1,* Christina Janko,1,* Marc Schwarz,2 László Dézsi,3 Rudolf Urbanics,4 Jasmin Matuszak,1 Erik Őrfi,3 Tamás Fülöp,3 Tobias Bäuerle,2 János Szebeni,3,4 Clément Journé,5 Aldo R Boccaccini,6 Christoph Alexiou,1 Stefan Lyer,1 Iwona Cicha1 1Cardiovascular Nanomedicine Unit, Section of Experimental Oncology und Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, ENT Department, University Hospital Erlangen, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 2Preclinical Imaging Platform Erlangen (PIPE), Institute of Radiology, University Hospital Erlangen, Erlangen, Germany; 3Nanomedicine Research and Education Center, Semmelweis University, 4SeroScience Ltd., Budapest, Hungary; 5Inserm U1148, Fédération de Recherche en Imagerie Multimodalités (FRIM), X Bichat Hospital, Paris Diderot University, Paris, France; 6Institute of Biomaterials, Department of Materials Science and Engineering, University Erlangen-Nuremberg, Erlangen, Germany *These authors contributed equally to this work Abstract: Iron oxide-based contrast agents have been in clinical use for magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system. Superparamagnetic iron oxide nanoparticles (SPIONs) have high potential as a contrast agent for MRI, but no intravenous iron oxide-containing agents are currently approved for clinical imaging. The aim of our work was to analyze the hemocompatibility and immuno-safety of a new type of dextran-coated SPIONs (SPIONdex) and to characterize these nanoparticles with ultra-high-field MRI. Key parameters related to nanoparticle hemocompatibility and immuno-safety were investigated in vitro and ex vivo. To address concerns associated with hypersensitivity reactions to injectable nanoparticulate agents, we analyzed complement activation-related pseudoallergy (CARPA) upon intravenous administration of SPIONdex in a pig model. Furthermore, the size-tunability of SPIONdex and the effects of size reduction on their biocompatibility were investigated. In vitro, SPIONdex did not induce hemolysis, complement or platelet activation, plasma coagulation, or leukocyte procoagulant activity, and had no relevant effect on endothelial cell viability or endothelial–monocytic cell interactions. Furthermore, SPIONdex did not induce CARPA even upon intravenous administration of 5 mg Fe/kg in pigs. Upon SPIONdex administration in mice, decreased liver signal intensity was observed after 15 minutes and was still detectable 24 h later. In addition, by changing synthesis parameters, a reduction in particle size

Ralf P Friedrich,1 Christina Janko,1 Marina Poettler,1 Philipp Tripal,1 Jan Zaloga,1 Iwona Cicha,1 Stephan Dürr,1,2 Johannes Nowak,3 Stefan Odenbach,3 Ioana Slabu,4 Maik Liebl,4 Lutz Trahms,4 Marcus Stapf,5 Ingrid Hilger,5 Stefan Lyer,1 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, 2Department of Otorhinolaryngology, Head and Neck Surgery, Section of Phoniatrics and Pediatric Audiology, University hospital Erlangen, Erlangen, 3Technische Universität Dresden, Chair of Magnetofluiddynamics, Measuring and Automation Technology, Dresden, 4Physikalisch-Technische Bundesanstalt Berlin, Berlin, 5Department of Radiology, Division of Diagnostic and Interventional Radiology, Experimental Radiology, University hospital Jena, Jena, Germany Abstract: Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of superparamagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. Keywords: low cytometry, side scatter, intracellular superparamagnetic iron oxide nanoparticles, quantification, spectroscopy

Jan Zaloga,1 Christina Janko,1 Johannes Nowak,2 Jasmin Matuszak,1 Sabine Knaup,1 Dietmar Eberbeck,3 Rainer Tietze,1 Harald Unterweger,1 Ralf P Friedrich,1 Stephan Duerr,1 Ralph Heimke-Brinck,4 Eva Baum,4 Iwona Cicha,1 Frank Dörje,4 Stefan Odenbach,2 Stefan Lyer,1 Geoffrey Lee,5 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section for Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, University Hospital Erlangen, Erlangen, Germany; 2Measuring and Automation Technology, Technical University Dresden, Dresden, Germany; 3Physikalisch-Technische-Bundesanstalt, Berlin, Germany; 4Pharmacy Department, University Hospital Erlangen, Erlangen, Germany; 5Division of Pharmaceutics, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany Abstract: The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 µg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical application. The synthesis is straightforward and reproducible and thus easily translatable into a good manufacturing practice environment. Keywords: iron oxide nanoparticles, drug delivery, protein corona, magnetic drug targeting, colloidal stability

Atherogenic diets rich in saturated fat and cholesterol influence the blood viscosity and red blood cell (RBC) aggregability, the parameters associated with increased risk of circulatory disorders. However, little is known about the effect of triglycerides, which are the major dietary lipid form in humans, on blood rheology. Therefore, we studied the effects of postprandial plasma triglyceride levels on human RBC indices, hematological parameters, RBCs aggregation velocity and whole blood viscosity. For this purpose, whole blood was collected 2 hours after high‐fat or low‐fat meal. Proteins, triglycerides and cholesterol levels of plasma were analysed, and RBCs rouleaux formation rate was measured in 70% autologous plasma using a low‐shear rheoscope. There were no significant differences in hematological parameters, RBC indices, whole blood viscosity, plasma protein and cholesterol content between high‐fat and low‐fat blood samples. However, a significant increase in rouleaux formation rate was observed in samples with high postprandial triglyceride levels, when compared with low‐triglyceride samples. Plasma triglyceride levels correlated significantly with rouleaux formation rate. In conclusion, these results suggest that diet‐dependent alterations of plasma triglyceride levels as well as possible changes in the cell membrane lipid composition lead to RBC hyperaggregability. [ABSTRACT FROM AUTHOR]

The rouleau formation of erythrocytes and the erythrocyte sedimentation were examined for mixture of different kinds of the cells suspended in isotonic phosphate-buffered saline containing 1 or 2 g/dl dextran T-70 (MW = 70,400) and 4 g/dl albumin, using a low shear rheoscope and the Westergren method, respectively. The deformability of cells was decreased by treating with diamide, diazene-dicarboxylic acid bis[N,N-dimethylamide], and the sialic acid content of cells, i.e., the surface negative charge, was reduced by treating with neuraminidase. (1) The rate of rouleau formation was decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. The rate changed in proportion to the ratio of the modified cells to normal cells. (2) The erythrocyte sedimentation was also decreased in cells with decreased deformability, while it was increased in cells with reduced sialic acid content. Furthermore, the erythrocyte sedimentation changed almost proportionally to the ratio of the modified cells to normal cells. (3) When normal deformable cells were mixed with cells with decreased deformability, the deformable cells seemed to settle faster than the less deformable cells, though the difference was not significant. (4) When normal cells were mixed with cells with reduced sialic acid content, the cells with less sialic acid settled significantly faster than those with more sialic acid. The present experiment may conclude that erythrocyte aggregation is induced preferentially among more deformable cells and/or among less negatively charged cells with weaker electrostatic repulsive force, and then the formed aggregates settle faster. [ABSTRACT FROM AUTHOR]

Atherosclerosis results from the accumulation of the modified lipoproteins within the artery walls, which triggers complex vascular inflammatory processes. Although the pharmacologic agents for the treatment of clinical manifestations of atherosclerosis are available, their systemic delivery has serious disadvantages, such as considerable side effects or low efficacy at tolerated doses. Moreover, the treatment of atherosclerosis using the interventional techniques bears further shortcomings: the implanted stents require a lengthy antiplatelet therapy and carry the risk of in-stent restenosis. In the surgical approach to atherosclerosis, apart from the overall risk of open heart surgery, the lack of adequate venous material for bypasses constitutes a common problem. The nanotechnology has the potential to overcome the disadvantages of the current therapy of atherosclerosis, e.g., by the formation of nanosized assemblies for the earlier detection of atherosclerotic lesions and for cell-specific delivery of therapeutics. Replacing the current systemic pharmacological approach by a locally targeted treatment of plaques can substantially minimize the adverse effects, by lowering the drug cytotoxicity and reducing the required dosage. Moreover, a new generation of nanotechnological approaches to the revascularization procedures is now emerging, e.g., vascular tissue engineering utilizing the magnetic nanoparticles or the design of stents with the reduced risk of thrombosis and restenosis. This review discusses the possible applications of the nanomedical approaches in the treatment of cardiovascular diseases.

The use of nanoparticles in tumor imaging, molecular imaging, and drug delivery has significantly expanded in the last few years. The relatively new field of “theranostics” combines their capacity for drug delivery with their potential as contrast agents. Depending on the imaging modality used, several types of nanoparticles are available, such as gold for optical imaging or superparamagnetic iron oxide for magnetic resonance imaging. This review will give a short overview of the different types of nanoparticles as well as their development and potential application in recent years. Furthermore, it describes the research on classic imaging modalities as well as on new techniques to image nanoparticles in vivo and focuses on magnetic-based imaging modalities.

In this study, fibrous polyurethane (PU) materials with average fiber diameter of 200, 500, and 1000 nm were produced using a solution blow spinning (SBS) process. The effects of the rotation speed of the collector (in the range of 200-25 000 rpm) on the fiber alignment and diameter were investigated. The results showed that fiber alignment was influenced by the rotation speed of the collector, and such alignment was possible when the fiber diameter was within a specific range. Homogeneously oriented fibers were obtained only for a fiber diameter ≥500 nm. Moreover, the changes in fiber orientation and fiber diameter (resulting from changes in the rotation speed of the collector) were more noticeable for materials with an average fiber diameter of 1000 nm in comparison to 500 nm, which suggests that the larger the fiber diameter, the better the controlled architectures that can be obtained. The porosity of the produced scaffolds was about 65-70%, except for materials with a fiber diameter of 1000 nm and aligned fibers, which had a higher porosity (76%). Thus, the scaffold pore size increased with increasing fiber diameter but decreased with increasing fiber alignment. The mechanical properties of fibrous materials strongly depend on the direction of stretching, whereby the fiber orientation influences the mechanical strength only for materials with a fiber diameter of 1000 nm. Furthermore, the fiber diameter and alignment affected the pericyte growth. Significant differences in cell growth were observed after 7 days of cell culture between materials with a fiber diameter of 1000 nm (cell coverage 96-99%) and those with a fiber diameter of 500 nm (cell coverage 70-90%). By appropriately setting the SBS process parameters, scaffolds can be easily adapted to the cell requirements, which is of great importance in producing complex 3D structures for guided tissue regeneration.

Cold water immersion (CWI) involves rapid cooling of the body, which, in healthy individuals, triggers a defence response to an extreme stimulus, to which the body reacts with stress. The aim of the study was to determine the effect of CWI on hemorheological blood indicators. The study group consisted of 13 young males. Blood samples were collected before and after CWI. The assessed parameters included the complete blood count, fibrinogen, hs-C-reactive protein (CRP), proteinogram, and blood rheology factors, such as erythrocyte elongation index (EI), half-time of total aggregation, and aggregation index. Additionally, the effect of reduced temperature on primary human vascular endothelium was investigated in vitro. CWI resulted in the decrease of body temperature to 31.55 ± 2.87 °C. After CWI, neutrophil count and mean corpuscular volume (MCV) were significantly increased in the study group, while lymphocyte count was significantly decreased. Significantly higher levels of total blood protein and albumin concentration were detected after the immersion. Among hemorheological characteristics, erythrocyte EIs at shear stress values ranging from 2.19 to 60.30 Pa were significantly lower after CWI. No significant changes in other rheological, morphological or biochemical parameters were observed. In vitro, human umbilical vein endothelial cells responded to 3 h of temperature decrease to 25 °C with unchanged viability, but increased recruitment of THP-1 monocytic cells and changes in cell morphology were observed. This was the first study to evaluate the effect of single CWI on rheological properties of blood in healthy young men. The results indicate that a single CWI may increase blood protein concentrations and worsen erythrocyte deformability parameters., (© 2024. The Author(s).)

Background: Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is β-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration., Methods: In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated., Results: Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI., Conclusions: The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future.

The traditional 3D culture systems in vitro lack the biological and mechanical spatiotemporal stimuli characteristic to native tissue development. In our study, we combined porous polysaccharide-based hydrogel scaffolds with a bioreactor-type perfusion device that generates favorable mechanical stresses while enhancing nutrient transfers. MC3T3E1 mouse osteoblasts were seeded in the scaffolds and cultivated for 3 weeks under dynamic conditions at a perfusion rate of 10 mL min -1 . The spatial distribution of the cells labeled with superparamagnetic iron oxide nanoparticles was visualized by MRI. Confocal microscopy was used to assess cell numbers, their distribution inside the scaffolds, cell viability, and proliferation. The oxygen diffusion coefficient in the hydrogel was measured experimentally. Numerical simulations of the flow and oxygen transport within the bioreactor were performed using a lattice Boltzmann method with a two-relaxation time scheme. Last, the influence of cell density and spheroid size on cell oxygenation was investigated. The cells spontaneously organized into spheroids with a diameter of 30-100 μm. Cell viability remained unchanged under dynamic conditions but decreased under static culture. The cell proliferation (Ki67 expression) in spheroids was not observed. The flow simulation showed that the local fluid velocity reached 27 mm s -1 at the height where the cross-sectional area of the flow was the smallest. The shear stress exerted by the fluid on the scaffolds may locally rise to 100 mPa, compared with the average value of 25 mPa. The oxygen diffusion coefficient in the hydrogel was 1.6×10-9 m 2 s -1 . The simulation of oxygen transport and consumption confirmed that the cells in spheroids did not suffer from hypoxia when the bioreactor was perfused at 10 mL min -1 , and suggested the existence of optimal spheroid size and spacing for appropriate oxygenation. Collectively, these findings enabled us to define the optimal conditions inside the bioreactor for an efficient in vitro cell organization and survival in spheroids, which are paramount to future applications with organoids.