Influence of Riboflavin Targeting on Tumor Accumulation and Internalization of Peptostar Based Drug Delivery Systems.

Riboflavin carrier protein (RCP) and riboflavin transporters (RFVTs) have been reported to be highly overexpressed in various cancer cells. Hence, targeting RCP and RFVTs using riboflavin may enhance tumor accumulation and internalization of drug delivery systems. To test this hypothesis, butyl-based 3-arm peptostar polymers were synthesized consisting of a lysine core (10 units per arm) and a sarcosine shell (100 units per arm). The end groups of the arms and the core were successfully modified with riboflavin and the Cy5.5 fluorescent dye, respectively. While in phosphate buffered saline the functionalized peptostars showed a bimodal behavior and formed supramolecular structures over time, they were stable in the serum maintaining their hydrodynamic diameter of 12 nm. Moreover, the polymers were biocompatible and the uptake of riboflavin targeted peptostars in A431 and PC3 cells was higher than in nontargeted controls and could be blocked competitively. In vivo, the polymers showed a moderate passive tumor accumulation, which was not significantly different between targeted and nontargeted peptostars. Nonetheless, at the histological level, internalization into tumor cells was strongly enhanced for the riboflavin-targeted peptostars. Based on these results, we conclude that passive accumulation is dominating the accumulation of peptostars, while tumor cell internalization is strongly promoted by riboflavin targeting.

Size-isolation of superparamagnetic iron oxide nanoparticles improves MRI, MPI and hyperthermia performance.

Superparamagnetic iron oxide nanoparticles (SPION) are extensively used for magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), as well as for magnetic fluid hyperthermia (MFH). We here describe a sequential centrifugation protocol to obtain SPION with well-defined sizes from a polydisperse SPION starting formulation, synthesized using the routinely employed co-precipitation technique. Transmission electron microscopy, dynamic light scattering and nanoparticle tracking analyses show that the SPION fractions obtained upon size-isolation are well-defined and almost monodisperse. MRI, MPI and MFH analyses demonstrate improved imaging and hyperthermia performance for size-isolated SPION as compared to the polydisperse starting mixture, as well as to commercial and clinically used iron oxide nanoparticle formulations, such as Resovist® and Sinerem®. The size-isolation protocol presented here may help to identify SPION with optimal properties for diagnostic, therapeutic and theranostic applications.

In vitro and in vivo evaluation of biohybrid tissue-engineered vascular grafts with transformative 1H/19F MRI traceable scaffolds.

Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive non-invasive imaging approaches to longitudinally monitor their functionality, integrity, and positioning. Here, we present an imaging approach comprising the labeling of non-degradable and degradable TEVGs' components for their in vitro and in vivo monitoring by hybrid 1H/19F MRI. TEVGs (inner diameter 1.5 mm) consisted of biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers passively incorporating superparamagnetic iron oxide nanoparticles (SPIONs), non-degradable polyvinylidene fluoride scaffolds labeled with highly fluorinated thermoplastic polyurethane (19F-TPU) fibers, a smooth muscle cells containing fibrin blend, and endothelial cells. 1H/19F MRI of TEVGs in bioreactors, and after subcutaneous and infrarenal implantation in rats, revealed that PLGA degradation could be faithfully monitored by the decreasing SPIONs signal. The 19F signal of 19F-TPU remained constant over weeks. PLGA degradation was compensated by cells' collagen and α-smooth-muscle-actin deposition. Interestingly, only TEVGs implanted on the abdominal aorta contained elastin. XTT and histology proved that our imaging markers did not influence extracellular matrix deposition and host immune reaction. This concept of non-invasive longitudinal assessment of cardiovascular implants using 1H/19F MRI might be applicable to various biohybrid tissue-engineered implants, facilitating their clinical translation.

Magnetic resonance imaging contrast-enhancement with superparamagnetic iron oxide nanoparticles amplifies macrophage foam cell apoptosis in human and murine atherosclerosis.

AIMS: (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidaemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidaemic models of atherosclerosis. METHODS AND RESULTS: RAW264.7 foam cells, exposed in vitro to ferumoxide (dextran-coated SPIO), ferumoxtran (dextran-coated USPIO), or ferumoxytol [carboxymethyl (CM) dextran-coated USPIO] (all 1 mg Fe/mL) showed increased apoptosis and reactive oxygen species accumulation for ferumoxide and ferumoxtran, whereas ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of ferumoxide (0.3 mg Fe/kg) and ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidaemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared with saline-treated controls. Again, ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P < 0.001, n = 8/group). Repeated ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed five-fold increased apoptosis (18.2 vs. 3.7%, respectively; P = 0.004) compared with controls who did not receive ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of ferumoxide and ferumoxtran. CONCLUSIONS: Ferumoxide and ferumoxtran, but not ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.

Monitoring the Remodeling of Biohybrid Tissue-Engineered Vascular Grafts by Multimodal Molecular Imaging.

Tissue-engineered vascular grafts (TEVGs) with the ability to grow and remodel open new perspectives for cardiovascular surgery. Equipping TEVGs with synthetic polymers and biological components provides a good compromise between high structural stability and biological adaptability. However, imaging approaches to control grafts' structural integrity, physiological function, and remodeling during the entire transition between late in vitro maturation and early in vivo engraftment are mandatory for clinical implementation. Thus, a comprehensive molecular imaging concept using magnetic resonance imaging (MRI) and ultrasound (US) to monitor textile scaffold resorption, extracellular matrix (ECM) remodeling, and endothelial integrity in TEVGs is presented here. Superparamagnetic iron-oxide nanoparticles (SPION) incorporated in biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers of the TEVGs allow to quantitatively monitor scaffold resorption via MRI both in vitro and in vivo. Additionally, ECM formation can be depicted by molecular MRI using elastin- and collagen-targeted probes. Finally, molecular US of αv ß3 integrins confirms the absence of endothelial dysfunction; the latter is provocable by TNF-α. In conclusion, the successful employment of noninvasive molecular imaging to longitudinally evaluate TEVGs remodeling is demonstrated. This approach may foster its translation from in vitro quality control assessment to in vivo applications to ensure proper prostheses engraftment.

Image-derived mean velocity measurement for prediction of coronary flow reserve in a canonical stenosis phantom using magnetic particle imaging.

INTRODUCTION: Aim of this study is to evaluate whether magnetic particle imaging (MPI) is capable of measuring velocities occurring in the coronary arteries and to compute coronary flow reserve (CFR) in a canonical phantom as a preliminary study. METHODS: For basic velocity measurements, a circulation phantom was designed containing replaceable glass tubes with three varying inner diameters, matching coronary-vessel diameters. Standardised boluses of superparamagnetic-iron-oxide-nanoparticles were injected and visualised by MPI. Two image-based techniques were competitively applied to calibrate the respective glass tube and to compute the mean velocity: full-duration-at-half-maximum (FDHM) and tracer dilution (TD) method. For CFR-calculation, four necessary settings of the circulation model of a virtual vessel with an inner diameter of 4 mm were generated using differently sized glass tubes and a stenosis model. The respective velocities in stenotic glass tubes were computed without recalibration. RESULTS: On velocity level, comparison showed a good agreement (rFDHM = 0.869, rTD = 0.796) between techniques, preferably better for 4 mm and 6 mm inner diameter glass tubes. On CFR level MPI-derived CFR-prediction performed considerably inferior with a relative error of 20-44%. CONCLUSIONS: MPI has the ability to reliably measure coronary blood velocities at rest as well as under hyperaemia and therefore may be suitable for CFR calculation. Calibration-associated accuracy of CFR-measurements has to be improved substantially in further studies.

Molecular magnetic resonance imaging of Alpha-v-Beta-3 integrin expression in tumors with ultrasound microbubbles.

Microbubbles (MB) are used as ultrasound (US) contrast agents and can be efficiently targeted against markers of angiogenesis and inflammation. Due to their gas core, MB locally alter susceptibilities in magnetic resonance imaging (MRI), but unfortunately, the resulting contrast is low and not sufficient to generate powerful molecular MRI probes. Therefore, we investigated whether a potent molecular MR agent can be generated by encapsulating superparamagnetic iron oxide nanoparticles (SPION) in the polymeric shell of poly (n-butylcyanoacrylate) (PBCA) MB and targeted them against αvß3 integrins on the angiogenic vasculature of 4T1 murine breast carcinomas. SPION-MB consist of an air core and a multi-layered polymeric shell enabling efficient entrapment of SPION. The mean size of SPION-MB was 1.61 ± 0.32 µm. Biotin-streptavidin coupling was employed to functionalize the SPION-MB with cyclic RGDfK (Arg-Gly-Asp) and RADfK (Arg-Ala-Asp) peptides. Cells incubated with RGD-SPION-MB showed enhanced transverse relaxation rates compared with SPION-MB and blocking αvß3 integrin receptors with excess free cRGDfK significantly reduced RGD-SPION-MB binding. Due to the fast binding of RGD-SPION-MB in vivo, dynamic susceptibility contrast MRI was employed to track their retention in tumors in real-time. Higher retention of RGD-SPION-MB was observed compared with SPION-MB and RAD-SPION-MB. To corroborate our MRI results, molecular US was performed the following day using the destruction-replenishment method. Both imaging modalities consistently indicated higher retention of RGD-SPION-MB in angiogenic vessels compared with SPION-MB and RAD-SPION-MB. Competitive blocking experiments in mice further confirmed that the binding of RGD-SPION-MB to αvß3 integrin receptors is specific. Overall, this study demonstrates that RGD-SPION-MB can be employed as molecular MR/US contrast agents and are capable of assessing the αvß3 integrin expression in the neovasculature of malignant tumors.

Novel dual-responsive semi-interpenetrating polymer network hydrogels for controlled release of anticancer drugs.

Novel dual-responsive hydrogels of semi-interpenetrating polymer networks (semi-IPNs) based on temperature-sensitive N-isopropylacrylamide (NIPAA) and pH-sensitive N-ethylmaleamic acid (NEMA) monomers and sodium alginate polymer were synthesized using free-radical polymerization in the presence of N,N'-methylenebisacrylamide as a crosslinker. Stimuli-responsive properties of the semi-IPN hydrogels showed remarkable sensitivity to both temperature and pH without limitation in NEMA content. Doxorubicin hydrochloride (DOX) as a model drug was efficiently loaded into the hydrogels and the release profiles of drug from them were investigated. The results of in vitro studies showed a quick DOX release in the conditions simulating tumor environment (phosphate-buffered saline [PBS], pH 6.5, 37°C) or endosomes/lysosomes (PBS, pH 5.5, 37°C) compared to simulated human physiological conditions (PBS, pH 7.4, 37°C). In conclusion, the novel poly(NIPAA-co-NEMA) semi-IPN hydrogels could be a promising candidate for targeted and controlled release of anticancer drugs in drug delivery system.

Iron oxide nanoparticles: Diagnostic, therapeutic and theranostic applications.

Many different iron oxide nanoparticles have been evaluated over the years, for a wide variety of biomedical applications. We here summarize the synthesis, surface functionalization and characterization of iron oxide nanoparticles, as well as their (pre-) clinical use in diagnostic, therapeutic and theranostic settings. Diagnostic applications include liver, lymph node, inflammation and vascular imaging, employing mostly magnetic resonance imaging but recently also magnetic particle imaging. Therapeutic applications encompass iron supplementation in anemia and advanced cancer treatments, such as modulation of macrophage polarization, magnetic fluid hyperthermia and magnetic drug targeting. Because of their properties, iron oxide nanoparticles are particularly useful for theranostic purposes. Examples of such setups, in which diagnosis and therapy are intimately combined and in which iron oxide nanoparticles are used, are image-guided drug delivery, image-guided and microbubble-mediated opening of the blood-brain barrier, and theranostic tissue engineering. Together, these directions highlight the versatility and the broad applicability of iron oxide nanoparticles, and indicate the integration in future medical practice of multiple iron oxide nanoparticle-based materials.