Fucoidan-coated coils improve healing in a rabbit elastase aneurysm model.

BACKGROUND: Recanalization of coiled aneurysms remains unresolved. To limit aneurysm recanalization after embolization with coils, we propose an innovative approach to optimize aneurysm healing using fucoidan-coated coils. OBJECTIVE: To evaluate the short-term efficacy and long-term safety of the new coil system with conventional angiography, histology, and multiphoton microscopy for follow-up of fibrosis and neointima formation. METHODS: We conducted a feasibility study on rabbit elastase-induced aneurysms. Embolization was carried out with bare platinum coils, fucoidan-coated coils, or dextran-coated coils. Aneurysms were controlled after 1 month by digital subtraction angiography (DSA). Aneurysm samples were collected and processed for histological analysis. Aneurysm healing and fibrosis were measured by quantifying collagen according to the histological healing score by combining standard light microscopy and multiphoton imaging. We divided 27 rabbits into three groups: bare platinum group, fucoidan group, and dextran group as controls. RESULTS: Angiographic grading showed a trend toward less recanalization in the fucoidan group, although there were no significant differences among the three groups (P=0.21). Histological healing was significantly different according to the presence of more collagen in the neck area of aneurysms in the fucoidan group versus the bare platinum group (P=0.011), but not in the dextran group. Histological index was significantly better at the aneurysm neck in the fucoidan group than in the bare platinum group (P=0.004). Collagen organization index was also significantly better in the fucoidan group than in the bare platinum group (P=0.007). CONCLUSION: This proof-of-concept study demonstrated the feasibility and efficacy of treatment with fucoidan-coated coils to improve aneurysm healing. The results in this rabbit in vivo model showed that fucoidan-coated coils have the potential to improve healing following endovascular treatment.

P-selectin targeting polysaccharide-based nanogels for miRNA delivery.

Nanogels were prepared in aqueous media without the use of any organic solvent via a simple polyelectrolyte complexation method between aminated pullulan and fucoidan followed by covalent crosslinking with genipin. Homogeneously distributed genipin crosslinked nanogels (G-PECs) were obtained with a mean hydrodynamic diameter of ~155 nm and zeta potential of 0.86 ± 4.35 mV. Their capacity to bind to human activated platelets was evaluated in vitro, as well as their cytocompatibility within human endothelial cells after 1 day of incubation up to 1000 µg/mL of G-PECs (94.56 ± 7.82% of viable cells). Additional hemolysis tests support the biocompatible character of the developed nanosystems (hemolysis rate of 2.09 ± 0.06% for 1000 µg/mL of G-PECs). Under acid conditions, the surface charge of G-PECs was tuned to around ~10 mV allowing miRNA incorporation via electrostatic interactions. G-PECs were able to promote miRNA delivery inside cells, as demonstrated by fluorescence microscopy images of labelled miRNA. With further studies to demonstrate the biological activity of delivered miRNA, these nanogels could be an interesting platform for miRNA-based therapeutics in atherothrombotic-related diseases thanks to the possibility to target over-expressed P-selectin.

Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases.

The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 µg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.

Amino-Fucoidan as a Vector for rtPA-Induced Fibrinolysis in Experimental Thrombotic Events.

Acute ischaemic stroke, myocardial infarction and pulmonary embolism are the main causes of mortality and morbidity worldwide. Thrombolysis by intravenous injection of recombinant tissue plasminogen activator (rtPA) remains the most common non-interventional treatment to recanalize occluded vessels. However, this procedure is limited by significant drawbacks, including high doses and bleeding complications. Recent studies showed that fucoidan targets the intraluminal thrombus in vivo. We have developed a chimaera covalently linking fucoidan, able to target platelets within the thrombus, to dilysine, able to non-covalently bind rtPA. We hypothesize that this construct should vectorize rtPA to the thrombus, thus increasing its fibrinolytic efficacy and avoiding its deleterious effects. In vitro, rtPA mixed to dilysine fucoidan (DLF) shows a greater fibrinolytic effect than rtPA alone, both on platelet-rich thrombus and in whole blood. In vivo, occluded mesenteric vessels, carotid artery and vena cava were more efficiently recanalized by DLF complexed to rtPA than by rtPA alone. This study thus provides evidence that DLF may be a promising therapeutic tool to fight against acute thrombosis by enhancing rtPA fibrinolytic efficiency.

Gadolinium/terbium hybrid macromolecular complexes for bimodal imaging of atherothrombosis.

We developed a fluorescence imaging microscope system intended for the localization within artery slices of a gadolinium-based macromolecular biospecific magnetic resonance (MR) contrast agent used for the visualization of atherothrombosis. As the contrast agent is not initially fluorescent, we substitute some gadolinium ions for terbium ions to make them fluorescent while preserving their chemical characteristics. A long fluorescence emission time constant enables us to have a suitable signal-to-noise ratio, despite a low intensity, using pulsed illumination and time-gated imaging. Images of rat arteries show that the contrast agent is indeed localized on the specific regions of the tissues. We currently have a tool that allows us to understand and optimize the MR contrast agent.

Development of Polymer Microcapsules Functionalized with Fucoidan to Target P-Selectin Overexpressed in Cardiovascular Diseases.

New tools for molecular imaging and targeted therapy for cardiovascular diseases are still required. Herein, biodegradable microcapsules (MCs) made of polycyanoacrylate and polysaccharide and functionalized with fucoidan (Fuco-MCs) are designed as new carriers to target arterial thrombi overexpressing P-selectin. Physicochemical characterizations demonstrated that microcapsules have a core-shell structure and that fucoidan is present onto the surface of Fuco-MCs. Furthermore, their sizes range from 2 to 6 µm and they are stable on storage over 30 d at 4 °C. Flow cytometry experiments evidenced the binding of Fuco-MCs for human activated platelets as compared to MCs (mean fluorescence intensity: 12 008 vs. 9, p < 0.001) and its absence for nonactivated platelets (432). An in vitro flow adhesion assay showed high specific binding efficiency of Fuco-MCs to P-selectin and to activated platelet aggregates under arterial shear stress conditions. Moreover, both types of microcapsules reveal excellent compatibility with 3T3 cells in cytotoxicity assay. One hour after intravenous injection of microcapsules, histological analysis revealed that Fuco-MCs are localized in the rat abdominal aortic aneurysm thrombotic wall and that the binding in the healthy aorta is low. In conclusion, these microcapsules appear as promising carriers for targeting of tissues characterized by P-selectin overexpression and for their molecular imaging or treatment.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity.

Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.

Ultrasmall superparamagnetic iron oxide nanoparticles coated with fucoidan for molecular MRI of intraluminal thrombus.

AIM: We have designed ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles associated with fucoidan (USPOI-FUCO), a natural sulfated polysaccharide with high affinity for activated platelets, to visualize by MRI arterial thrombi. MATERIALS & METHODS: USPIOs were prepared and sizes, zeta-potentials and relaxivities were measured. Elastase perfusion in the infrarenal aorta of Wistar rats induced intraluminal thrombus. They were scanned on 4.7 T MRI before and after injection of USPIO-FUCO or USPIO coated with anionic dextran. RESULTS: Surface plasmon resonance evidenced that fucoidan and USPIO-FUCO bind in vitro to immobilized P-selectin. All intraluminal hyposignals detected by MRI after injection of USPIO-FUCO on animals (13 out of 13) were correlated by histology with thrombi, whereas none could be identified with control USPIOs (0 out of 7). No signal was seen in absence of thrombus. Thrombi by MRI were correlated with P-selectin immunostaining and USPIO detection by electron microscopy. CONCLUSION: In vivo thrombi can thus be evidenced by MRI with USPIO-FUCO.

Purification of a low molecular weight fucoidan for SPECT molecular imaging of myocardial infarction.

Fucoidans constitute a large family of sulfated polysaccharides with several biochemical properties. A commercial fucoidan from brown algae, containing low molecular weight polysaccharidic species constituted of l-fucose, uronic acids and sulfate groups, was simply treated here with calcium acetate solution. This treatment led to a purified fraction with a yield of 45%. The physicochemical characterizations of the purified fucoidan using colorimetric assay, MALLS, dRI, FT-IR, NMR, exhibited molecular weight distributions and chemical profiles similar for both fucoidans whereas the sulfate and l-fucose contents increased by 16% and 71%, respectively. The biodistribution study in rat of both compounds labeled with 99mTc evidenced a predominant renal elimination of the purified fucoidan, but the crude fucoidan was mainly retained in liver and spleen. In rat myocardial ischemia-reperfusion, we then demonstrated the better efficiency of the purified fucoidan. This purified sulfated polysaccharide appears promising for the development of molecular imaging in acute coronary syndrome.

Lipid oxidation in baked products: impact of formula and process on the generation of volatile compounds.

This paper investigates the effect of ingredients on the reactions occurring during the making of sponge cake and leading to the generation of volatile compounds related to flavour quality. To obtain systems sensitive to lipid oxidation (LO), a formulation design was applied varying the composition of fatty matter and eggs. Oxidation of polyunsaturated fatty acids (PUFA) and formation of related volatile compounds were followed at the different steps of cake-making. Optimised dynamic Solid Phase Micro Extraction was applied to selectively extract either volatile or semi-volatile compounds directly from the baking vapours. We show for the first time that in the case of alveolar baked products, lipid oxidation occurs very early during the step of dough preparation and to a minor extent during the baking process. The generation of lipid oxidation compounds depends on PUFA content and on the presence of endogenous antioxidants in the raw matter. Egg yolk seemed to play a double role on reactivity: protecting unsaturated lipids from oxidation and being necessary to generate a broad class of compounds of the Maillard reaction during baking and linked to the typical flavour of sponge cake.