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.

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.

Fucoidans in Nanomedicine.

Fucoidans are widespread cost-effective sulfated marine polysaccharides which have raised interest in the scientific community over last decades for their wide spectrum of bioactivities. Unsurprisingly, nanomedicine has grasped these compounds to develop innovative therapeutic and diagnostic nanosystems. The applications of fucoidans in nanomedicine as imaging agents, drug carriers or for their intrinsic properties are reviewed here after a short presentation of the main structural data and biological properties of fucoidans. The origin and the physicochemical specifications of fucoidans are summarized in order to discuss the strategy of fucoidan-containing nanosystems in Human health. Currently, there is a need for reproducible, well characterized fucoidan fractions to ensure significant progress.

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.

VCAM-1-targeting gold nanoshell probe for photoacoustic imaging of atherosclerotic plaque in mice.

The development of molecular probes and novel imaging modalities, allowing better resolution and specificity, is associated with an increased potential for molecular imaging of atherosclerotic plaques especially in basic and pre-clinical research applications. In that context, a photoacoustic molecular probe based on gold nanoshells targeting VCAM-1 in mice (immunonanoshells) was designed. The molecular probe was validated in vitro and in vivo, showing no noticeable acute toxic effects. We performed the conjugation of gold nanoshells displaying near-infrared absorption properties with VCAM-1 antibody molecules and PEG to increase their biocompatibility. The resulting immunonanoshells obtained under different conditions of conjugation were then assessed for specificity and sensitivity. Photoacoustic tomography was performed to determine the ability to distinguish gold nanoshells from blood both in phantoms and in vivo. Ex vivo optical projection tomography of hearts and aortas from atherosclerotic and control mice confirmed the selective accumulation of the immunonanoshells in atherosclerotic-prone regions in mice, thus validating the utility of the probe in vivo in small animals for pre-clinical research. These immunonanoshells represent an adequate mean to target atherosclerotic plaques in small animals, leading to new tools to follow the effect of therapies on the progression or regression of the disease.