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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

Biosensor-integrated drug delivery systems are innovative devices in the health area, enabling continuous monitoring and drug administration. The use of smart polymer, bioMEMS, and electrochemical sensors have been extensively studied for these systems, especially for chronic diseases such as diabetes mellitus, cancer and cardiovascular diseases as well as advances in regenerative medicine. Basically, the technology involves sensors designed for the continuous analysis of biological molecules followed by drug release in response to specific signals. The advantages include high sensitivity and fast drug release. In this work, the main advances of biosensor-integrated drug delivery systems as new biomedical materials to improve the patients’ quality of life with chronic diseases are discussed.

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.

A facile and flexible approach for the integration of biomimetically branched microvasculature within bulk hydrogels is presented. For this, sacrificial scaffolds of thermoresponsive poly(2-cyclopropyl-2-oxazoline) (PcycloPrOx) are created using melt electrowriting (MEW) in an optimized and predictable way and subsequently placed into a customized bioreactor system, which is then filled with a hydrogel precursor solution. The aqueous environment above the lower critical solution temperature (LCST) of PcycloPrOx at 25 °C swells the polymer without dissolving it, resulting in fusion of filaments that are deposited onto each other (print-and-fuse approach). Accordingly, an adequate printing pathway design results in generating physiological-like branchings and channel volumes that approximate Murray's law in the geometrical ratio between parent and daughter vessels. After gel formation, a temperature decrease below the LCST produces interconnected microchannels with distinct inlet and outlet regions. Initial placement of the sacrificial scaffolds in the bioreactors in a pre-defined manner directly yields perfusable structures via leakage-free fluid connections in a reproducible one-step procedure. Using this approach, rapid formation of a tight and biologically functional endothelial layer, as assessed not only through fluorescent dye diffusion, but also by tumor necrosis factor alpha (TNF-α) stimulation, is obtained within three days.

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

Rapid endothelialization helps overcome the limitations of small-diameter vascular grafts. To develop biomimetic non-thrombogenic coatings supporting endothelialization, medical-grade polyurethane (PU) nanofibrous mats and tubular scaffolds with a diameter below 6 mm prepared by solution blow spinning were coated with polydopamine (PDA), or PDA and gelatin (PDA/Gel). The scaffolds were characterized by scanning electron microscopy, porosity measurement, tensile testing, wettability, Fourier Transform Infrared spectroscopy, and termogravimetric analysis, followed by the measurement of coating stability on the tubular scaffolds. The effect of coating on scaffold endothelialization and hemocompatibility was evaluated using human umbilical vein endothelial cells (HUVECs) and human platelets, showing low numbers of adhering platelets and significantly higher numbers of HUVECs on PDA- and PDA/Gel-coated mats compared to control samples. Tubular PU scaffolds and commercial ePTFE prostheses coated with PDA or PDA/Gel were colonized with HUVECs using radial magnetic cell seeding. PDA/Gel-coated samples achieved full endothelial coverage within 1-3 days post-endothelialization. Altogether, PDA and PDA/Gel coating significantly enhance the endothelialization on the flat surfaces, tubular small-diameter scaffolds, and commercial vascular prostheses. The presented approach constitutes a fast and efficient method of improving scaffold colonization with endothelial cells, expected to work equally well upon implantation.

Cell-sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become nonadhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. Polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for block copolymers, physisorbed onto substrates in a monomolecular thin film to achieve this. Consequently, such substrates, and the polymers required for film formation, can only be prepared in a chemical lab with profound macromolecular expertise. In this study, we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt % aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells, and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.

Iron oxide nanoparticles are a promising platform for biomedical applications, both in terms of diagnostics and therapeutics. In addition, arginine-rich polypeptides are known to penetrate across cell membranes. Here, we thus introduce a system based on magnetite nanoparticles and the polypeptide poly-l-arginine (polyR-Fe3O4). We show that the hybrid nanoparticles exhibit a low cytotoxicity that is comparable to Resovist&reg, a commercially available drug. PolyR-Fe3O4 particles perform very well in diagnostic applications, such as magnetic particle imaging (1.7 and 1.35 higher signal respectively for the 3rd and 11th harmonic when compared to Resovist&reg, ), or as contrast agents for magnetic resonance imaging (R2/R1 ratio of 17 as compared to 11 at 0.94 T for Resovist&reg, ). Moreover, these novel particles can also be used for therapeutic purposes such as hyperthermia, achieving a specific heating power ratio of 208 W/g as compared to 83 W/g for Feridex&reg, another commercially available product. Therefore, we envision such materials to play a role in the future theranostic applications, where the arginine ability to deliver cargo into the cell can be coupled to the magnetite imaging properties and cancer fighting activity.

Composite biomaterials based on proteins and inorganic fillers are highly attractive for wound dressing applications due to their highly absorbent properties towards blood and exudate provided by the inorganic fillers. Moreover, such composites offer a desirable environment for cells due to the combination of organic and inorganic characteristics. This study highlights the fabrication of soy protein isolate/nanoscale bioactive glass composite films by solvent casting method as a matrix for wound-dressing applications. The effect of the addition of bioactive glass nano particles on blood clotting was assessed. Cytotoxicity and in vitro cytocompatibility of the films were also tested. The results showed that the composite films could meet the essential requirements for an appropriate wound dressing with additional favorable properties such as hemostatic capability and mechanical properties as well as significant cell cytocompatibility.

Cardiovascular diseases (CVD) account for nearly half of all deaths in Europe and almost 30% of global deaths. Despite the improved clinical management, cardiovascular mortality is predicted to rise in the next decades due to the increasing impact of aging, obesity, and diabetes. The goal of emerging cardiovascular nanomedicine is to reduce the burden of CVD using nanoscale medical products and devices. However, the development of novel multicom-ponent nano-sized products poses multiple technical, ethical, and regulatory challenges, which often obstruct their road to successful approval and use in clinical practice. This review discusses the rational design of nanoparticles, including safety considerations and regulatory issues, and highlights the steps needed to achieve efficient clinical translation of promising nanomedicinal products for cardiovascular applications.

Hydrogels from natural polymers are widely used in tissue engineering due to their unique properties, especially when regarding the cell environment and their morphological similarity to the extracellular matrix (ECM) of native tissues. In this study, we describe the production and characterization of novel hybrid hydrogels composed of alginate blended with elastin from bovine neck ligament. The properties of elastin as a component of the native ECM were combined with the excellent chemical and mechanical stability as well as biocompatibility of alginate to produce two hybrid hydrogels geometries, namely 2D films obtained using sonication treatment and 3D microcapsules produced by pressure-driven extrusion. The resulting blend hydrogels were submitted to an extensive physico-chemical characterization. Furthermore, the biological compatibility of these materials was assessed using normal human dermal fibroblasts, indicating the suitability of this blend for soft tissue engineering.

Fabrication of microchannels using 3D printing of sugars as fugitive material is explored in different fields, including microfluidics. However, establishing reproducible methods for the controlled production of sugar structures with sub-100 μm dimensions remains a challenge. This study pioneers the processing of sugars by melt electrowriting (MEW) enabling the fabrication of structures with so far unprecedented resolution from Isomalt. Based on a systematic variation of process parameters, fibers with diameters down to 20 μm can be fabricated. The flexibility in the adjustment of fiber diameter by on-demand alteration of MEW parameters enables generating constructs with perfusable channels within polydimethylsiloxane molds. These channels have a diameter that can be adjusted from 30 to 200 μm in a single design. Taken together, the experiments show that MEW strongly benefits from the thermal and physical stability of Isomalt, providing a robust platform for the fabrication of small-diameter embedded microchannel systems.

Tissue-engineered scaffolds require an effective colonization with cells. Superparamagnetic iron oxide nanoparticles (SPIONs) can enhance cell adhesion to matrices by magnetic cell seeding. We investigated the possibility of improving cell attachment and growth on different alginate-based hydrogels using fibroblasts and endothelial cells (ECs) loaded with SPIONs. Hydrogels containing pure alginate (Alg), alginate dialdehyde crosslinked with gelatin (ADA-G) and Alg blended with G or silk fibroin (SF) were prepared. Endothelial cells and fibroblasts loaded with SPIONs were seeded and grown on hydrogels for up to 7 days, in the presence of magnetic field during the first 24 h. Cell morphology (fluorescent staining) and metabolic activity (WST-8 assay) of magnetically-seeded versus conventionally seeded cells were compared. Magnetic seeding of ECs improved their initial attachment and further growth on Alg/G hydrogel surfaces. However, we did not achieve an efficient and stable colonization of ADA-G films with ECs even with magnetic cell seeding. Fibroblast showed good initial colonization and growth on ADA-G and on Alg/SF. This effect was further significantly enhanced by magnetic cell seeding. On pure Alg, initial attachment and spreading of magnetically-seeded cells was dramatically improved compared to conventionally-seeded cells, but the effect was transient and diminished gradually with the cessation of magnetic force. Our results demonstrate that magnetic seeding improves the strength and uniformity of initial cell attachment to hydrogel surface in cell-specific manner, which may play a decisive role for the outcome in tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2948-2956, 2017.

Nearly one fourth of all deaths worldwide are attributable to cardiovascular diseases (CVD), including stroke and ischemic heart disease. In spite of the steady progress in cardiovascular diagnostics and therapy, the CVD prevalence continues to rise on a global scale. Persisting efforts in the fields of bio-engineering and tissue engineering, in parallel with improved understanding of disease mechanisms, have shifted the focus of research towards improved approaches to vascular regeneration, based on degradable biomaterials for temporary rather than permanent grafting. Utilizing advanced bioplotting techniques in order to combine cells, natural hydrogels and fibers, the production of tailored 3D vessel constructs that promote the regeneration of native tubular tissues has now become achievable. In this paper, we review the most promising biomaterials and additive manufacturing approaches to fabricate novel vascular constructs. In addition, we discuss the progress and the remaining challenges in the field.

Magnetic drug targeting utilizes an external magnetic field to target superparamagnetic iron oxide nanoparticles (SPIONs) and their cargo to the diseased vasculature regions. In the arteries, the flow conditions affect the behavior of magnetic particles and the efficacy of their accumulation. In order to estimate the magnetic capture of SPIONs in more physiological-like settings, we previously established an ex vivo model based on human umbilical cord arteries. The artery model was employed in our present studies in order to analyze the effects of the blood components on the efficacy of magnetic targeting, utilizing 2 types of SPIONs with different physicochemical characteristics. In the presence of freshly isolated human plasma or whole blood, a strong increase in iron content measured by AES was observed for both particle types along the artery wall, in parallel with clotting activation due to endogenous thrombin generation in plasma. Subsequent studies therefore utilized SPION suspensions in serum and washed red blood cells (RBCs) at hematocrit 50%. Interestingly, in contrast to cell culture medium suspensions, magnetic accumulation of circulating SPION-3 under the external magnet was achieved in the presence of RBCs. Taken together, our data shows that the presence of blood components affects, but does not prevent, the magnetic accumulation of circulating SPIONs.

International audience; The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0-400 mu g/mL) on endothelial-monocytic cell interactions in response to TNF-alpha was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0-100 mu g/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0-400 mu g/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25-50 mu g/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60-70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.

A variety of novel therapeutic approaches presented by several trials (e.g., FOURIER [Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk]/ODYSSEY OUTCOMES [Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With

The global burden of atherothrombotic diseases continues to grow. Worldwide, 17.9 million deaths per year are caused by cardiovascular diseases (CVD). According to WHO estimates, this number is expected to grow to more than 23.6 million by 2030. To achieve a more effective primary prevention of cardiovascular morbidity and mortality, the development of new diagnostic and therapeutic strategies is required. Nanomedicine offers a unique platform for novel approaches to the diagnosis and therapy of CVD. In particular iron oxide-based magnetic nanoparticles, a class of nanosized agents with superparamagnetic properties, are considered a promising tool in management of cardiovascular patients. Their possible applications range from plaque imaging and thrombus detection to the targeted drug-delivery, stent endothelialisation and blood vessel regeneration. This paper summarizes recent advances in the implementation of magnetic nanoparticles to diagnosis and treatment of cardiovascular disorders.

Due to the presence of tripeptide arginine–glycine–aspartic acid, gelatin is considered a very promising additive material to improve the cytocompatibility of alginate-based hydrogels. Two different strategies, physical blending and chemical crosslinking with gelatin, are used in this study to modify alginate hydrogel. As the intermolecular interactions between the polysaccharide and protein in the resulting physically blended and chemically crosslinked hydrogels are different, significant differences in the properties of these hydrogel types, regarding especially their surface topography, degradation kinetics, mechanical properties, and protein release behavior, are observed. Cellular behavior on both types of alginate–gelatin hydrogels is investigated using primary human dermal fibroblasts to elucidate the effects of the different structural, mechanical, and degradation properties of the produced hydrogels on fibroblast attachment and growth. The hydrogel that is chemically crosslinked with gelatin exhibits the highest degree of cytocompatibility regarding adhesion, proliferation, metabolic activity, and morphology of growing fibroblasts.

Recent years have brought about substantial advances in nanomedical approaches to human diseases. Novel thera- peutic and diagnostic nanoparticulate agents can be delivered via several routes, including enteral, transdermal, inhalational, as well as parenteral application. For nanoparticles administered via intravascular route, endothelial cells represent the first- contact cells and their responses to the candidate nanosystems should be considered before clinical applications. Additionally, a number of drug-delivery nanosystems that target endothelium are currently proposed. It is increasingly evident that the future progress in the treatment of many human maladies, including cardiovascular diseases and cancer, will be closely bound with the development of endothelium-targeting nanosystems. As endothelial cells in circulation are constantly exposed to shear stress, investigating nanoparticle-endothelial interactions under flow conditions is necessary to estimate the cell responses in physiological-like settings. This mini-review focuses on the recently reported studies assessing the uptake of circulating nanoparticles and their biological effects on endothelial cells, and summarizes the targeting approaches to enhance endothelial internalization of nanoparticles under flow conditions.

Surface-functionalized gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) may be a useful tool in various biomedical applications. To obtain Au-SPIONs, gold salt was precipitated onto citrate-stabilized SPIONs (Cit-SPIONs) using a simple, aqueous one-pot technique inspired by the Turkevich method of gold nanoparticle synthesis. By the further stabilization of the Au-SPION surface with additional citrate (Cit-Au-SPIONs), controllable and reproducible Z-averages enhanced long-term dispersion stability and moderate dispersion pH values were achieved. The citrate concentration of the reaction solution and the gold/iron ratio was found to have a major influence on the particle characteristics. While the gold-coating reduced the saturation magnetization to 40.7% in comparison to pure Cit-SPIONs, the superparamagnetic behavior of Cit-Au-SPIONs was maintained. The formation of nanosized gold on the SPION surface was confirmed by X-ray diffraction measurements. Cit-Au-SPION concentrations of up to 100 &micro, g Fe/mL for 48 h had no cytotoxic effect on Jurkat cells. At a particle concentration of 100 &micro, g Fe/mL, Jurkat cells were found to take up Cit-Au-SPIONs after 24 h of incubation. A significantly higher attachment of thiol-containing L-cysteine to the particle surface was observed for Cit-Au-SPIONs (53%) in comparison to pure Cit-SPIONs (7%).

The availability of cell growth-supporting materials for regenerative medicine is limited. Here, we aimed to improve the attachment and growth of endothelial cells (HUVECs) and fibroblasts on electrospun poly(e-caprolactone) (PCL) mats with different fiber diameters and on newly-established PCL and silk fibroin (PCL/SF) blended fibers obtained by using benign solvents (mixture of acetic acid and formic acid) for electrospinning. PCL mats were produced in two different average fiber diameters, namely micro and nano range. The electrospinning method was optimized to produce PCL/SF blended fibers. Morphology (F-actin), viability (calcein) and metabolic activity (WST-8 assay) of HUVECs and fibroblasts cultured in 2D on the mats of different fiber size and composition were compared. Subsequently, cylindrical PCL/SF scaffolds were produced and colonized with cells using 3D radial magnetic cell seeding. Both PCL and PCL/SF nanofibers provided better support for initial cell adhesion (day 1) compared with PCL microfibers. At day 7, the highest metabolic activity in HUVECs and fibroblasts was observed on PCL/SF mats. Microscopic studies confirmed that on day 7, the best support for cell growth was observed on PCL/SF. Tubular PCL/SF scaffolds were successfully colonized using magnetic nanoparticles and 3D radial magnetic cell seeding. Taken together, the fibers obtained by blending SF with PCL combine the mechanical benefits of PCL with improved biological functionality of SF. Electrospun PCL/SF nanofibers represent a promising material to produce both flat and 3D structures with potential for enhanced cell attachment and tissue regeneration.