Hyaluronic acid from bluefin tuna by-product: Structural analysis and pharmacological activities.

The fishing and aquaculture industries generate a huge amount of waste during processing and preservation operations, especially those of tuna. Recovering these by-products is a major economic and environmental challenge for manufacturers seeking to produce new active biomolecules of interest. A new hyaluronic acid was extracted from bluefin tuna's vitreous humour to assess its antioxidant and pharmacological activities. The characterization by infrared spectroscopy (FT-IR), nuclear magnetic resonance ((1D1H) and 2D (1H COSY, 1H/13C HSQC)) and size exclusion chromatography (SEC/MALS/DRI/VD) revealed that the extracted polysaccharide was a hyaluronic acid with high uronic acid content (55.8 %) and a weight average molecular weight of 888 kDa. This polymer possesses significant anti-radical activity and ferrous chelating capacity. In addition, pharmacological evaluation of its anti-inflammatory and analgesic potential, using preclinical models, in comparison with reference drugs (Dexamethasone, diclofenac, and acetylsalicylate of lysine), revealed promising anti-inflammatory activity as well as interesting peripheral and central antinociceptive activity. Therefore, our new hyaluronic acid compound may therefore serve as a potential drug candidate for the treatment of pain sensation and inflammation of various pathological origins.

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.

Spatial-Controlled Coating of Pro-Angiogenic Proteins on 3D Porous Hydrogels Guides Endothelial Cell Behavior.

In tissue engineering, the composition and the structural arrangement of molecular components within the extracellular matrix (ECM) determine the physical and biochemical features of a scaffold, which consequently modulate cell behavior and function. The microenvironment of the ECM plays a fundamental role in regulating angiogenesis. Numerous strategies in tissue engineering have attempted to control the spatial cues mimicking in vivo angiogenesis by using simplified systems. The aim of this study was to develop 3D porous crosslinked hydrogels with different spatial presentation of pro-angiogenic molecules to guide endothelial cell (EC) behavior. Hydrogels with pores and preformed microchannels were made with pharmaceutical-grade pullulan and dextran and functionalized with novel pro-angiogenic protein polymers (Caf1-YIGSR and Caf1-VEGF). Hydrogel functionalization was achieved by electrostatic interactions via incorporation of diethylaminoethyl (DEAE)-dextran. Spatial-controlled coating of hydrogels was realized through a combination of freeze-drying and physical absorption with Caf1 molecules. Cells in functionalized scaffolds survived, adhered, and proliferated over seven days. When incorporated alone, Caf1-YIGSR mainly induced cell adhesion and proliferation, whereas Caf1-VEGF promoted cell migration and sprouting. Most importantly, directed cell migration required the presence of both proteins in the microchannel and in the pores, highlighting the need for an adhesive substrate provided by Caf1-YIGSR for Caf1-VEGF to be effective. This study demonstrates the ability to guide EC behavior through spatial control of pro-angiogenic cues for the study of pro-angiogenic signals in 3D and to develop pro-angiogenic implantable materials.

Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

miRNA Delivery by Nanosystems: State of the Art and Perspectives.

MicroRNAs (miRNAs) are short (~21-23 nucleotides), non-coding endogenous RNA molecules that modulate gene expression at the post-transcriptional level via the endogenous RNA interference machinery of the cell. They have emerged as potential biopharmaceuticals candidates for the treatment of various diseases, including cancer, cardiovascular and metabolic diseases. However, in order to advance miRNAs therapeutics into clinical settings, their delivery remains a major challenge. Different types of vectors have been investigated to allow the delivery of miRNA in the diseased tissue. In particular, non-viral delivery systems have shown important advantages such as versatility, low cost, easy fabrication and low immunogenicity. Here, we present a general overview of the main types of non-viral vectors developed for miRNA delivery, with their advantages, limitations and future perspectives.

Fucoidan-functionalized polysaccharide submicroparticles loaded with alteplase for efficient targeted thrombolytic therapy.

Intravenous administration of fibrinolytic drugs is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and might trigger hemorrhagic transformations. Therefore, it is mandatory to develop innovative nanomedicine-based solutions for more efficient and safer thrombolysis with biocompatible and biodegradable thrombus-targeted nanocarrier. Herein, fucoidan-functionalized hydrogel polysaccharide submicroparticles with high biocompatibility are elaborated by the inverse miniemulsion/crosslinking method. They are loaded with the gold standard fibrinolytic - alteplase - to direct site-specific fibrinolysis due to nanomolar interactions between fucoidan and P-selectin overexpressed on activated platelets and endothelial cells in the thrombus area. The thrombus targeting properties of these particles are validated in a microfluidic assay containing recombinant P-selectin and activated platelets under arterial and venous blood shear rates as well as in vivo. The experiments on the murine model of acute thromboembolic ischemic stroke support this product's therapeutic efficacy, revealing a faster recanalization rate in the middle cerebral artery than with free alteplase, which reduces post-ischemic cerebral infarct lesions and blood-brain barrier permeability. Altogether, this proof-of-concept study demonstrates the potential of a biomaterial-based targeted nanomedicine for the precise treatment of acute thrombotic events, such as ischemic stroke.

Characterization of dextran particle size: How frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) coupled online with dynamic light scattering (DLS) leads to enhanced size distribution.

Accurate determinations of particle size and particle size distribution (PSD) are essential to achieve the clinical translation of medical nanoparticles (NPs). Herein, dextran-based NPs produced via a water-in-oil emulsification/crosslinking process and developed as nanomedicines were studied. NPs were first characterized using traditional batch-mode techniques as dynamic light scattering (DLS) and laser diffraction. In a second step, their analysis by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) was explored. The major parameters of the AF4 procedure, namely, crossflow, detector flow, crossflow decay programming and relaxation time were set up. The sizes of the particle fractions eluted under optimized conditions were measured using DLS as an online detector. We demonstrate that FI-AF4 is a powerful method to characterize dextran-NPs in the 200 nm -1 µm range. It provided a more realistic and comprehensive picture of PSD, revealing its heterogenous character and clearly showing the ratio of different populations in the sample, while batch-mode light scattering techniques only detected the biggest particle sizes.

Bioactive refinement for endosaccular treatment of intracranial aneurysms.

Endovascular treatment is the first-line therapy for most intracranial aneurysms; however, recanalisation remains a major limitation. Developments in bioengineering and material science have led to a novel generation of coil technologies for aneurysm embolisation that address clinical challenges of aneurysm recurrence. This review presents an overview of modified surface coil technologies and summarises the state of the art regarding their efficacy and limitations based on experimental and clinical results. We also present potential perspectives to develop biologically optimised devices.

Coronary stent CD31-mimetic coating favours endothelialization and reduces local inflammation and neointimal development in vivo.

AIMS: The rapid endothelialization of bare metal stents (BMS) is counterbalanced by inflammation-induced neointimal growth. Drug-eluting stents (DES) prevent leukocyte activation but impair endothelialization, delaying effective device integration into arterial walls. Previously, we have shown that engaging the vascular CD31 co-receptor is crucial for endothelial and leukocyte homeostasis and arterial healing. Furthermore, we have shown that a soluble synthetic peptide (known as P8RI) acts like a CD31 agonist. The aim of this study was to evaluate the effect of CD31-mimetic metal stent coating on the in vitro adherence of endothelial cells (ECs) and blood elements and the in vivo strut coverage and neointimal growth. METHODS AND RESULTS: We produced Cobalt Chromium discs and stents coated with a CD31-mimetic peptide through two procedures, plasma amination or dip-coating, both yielding comparable results. We found that CD31-mimetic discs significantly reduced the extent of primary human coronary artery EC and blood platelet/leukocyte activation in vitro. In vivo, CD31-mimetic stent properties were compared with those of DES and BMS by coronarography and microscopy at 7 and 28 days post-implantation in pig coronary arteries (n = 9 stents/group/timepoint). Seven days post-implantation, only CD31-mimetic struts were fully endothelialized with no activated platelets/leukocytes. At day 28, neointima development over CD31-mimetic stents was significantly reduced compared to BMS, appearing as a normal arterial media with the absence of thrombosis contrary to DES. CONCLUSION: CD31-mimetic coating favours vascular homeostasis and arterial wall healing, preventing in-stent stenosis and thrombosis. Hence, such coatings seem to improve the metal stent biocompatibility.

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.

Gd(DOTA)-grafted submicronic polysaccharide-based particles functionalized with fucoidan as potential MR contrast agent able to target human activated platelets.

Early detection of thrombotic events remains a big medical challenge. Dextran-based submicronic particles bearing Gd(DOTA) groups and functionalized with fucoidan have been produced via a simple and green water-in-oil emulsification/co-crosslinking process. Their capacity to bind to human activated platelets was evidenced in vitro as well as their cytocompatibility with human endothelial cells. The presence of Gd(DOTA) moieties was confirmed by elemental analysis and total reflection X-ray fluorescence (TRXF) spectrometry. Detailed characterization of particles was performed in terms of size distribution, morphology, and relaxation rates. In particular, longitudinal and transversal proton relaxivities were respectively 1.7 and 5.0 times higher than those of DOTAREM. This study highlights their potential as an MRI diagnostic platform for atherothrombosis.

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery.

Pullulan is a natural polysaccharide of potential interest for biomedical applications due to its non-toxic, non-immunogenic and biodegradable properties. The aim of this work was to synthesize cationic pullulan derivatives able to form complexes with microRNAs (miRNAs) driven by electrostatic interaction (polyplexes). Quaternized ammonium groups were linked to pullulan backbone by adding the reactive glycidyltrimethylammonium chloride (GTMAC). The presence of these cationic groups within the pullulan was confirmed by elemental analysis, Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The alkylated pullulan was able to interact with miRNA and form stable polyplexes that were characterized regarding size, zeta potential and morphology. The presence of miRNA was confirmed by agarose gel electrophoresis and UV spectrophotometry. In vitro tests on human umbilical vein endothelial cells did not show any cytotoxicity after 1 day of incubation with nanosized polyplexes up to 200 µg/mL. QA-pullulan was able to promote miRNA delivery inside cells as demonstrated by fluorescence microscopy images of labelled miRNA. In conclusion, the formation of polyplexes using cationic derivatives of pullulan with miRNA provided an easy and versatile method for polysaccharide nanoparticle production in aqueous media and could be a new promising platform for gene delivery.

Early Detection of Localized Immunity in Experimental Autoimmune Myocarditis Using [99mTc]Fucoidan SPECT.

PURPOSE: The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([99mTc]fucoidan), a molecular imaging agent specific for selectins, in the assessment of early localized immunity in a rat model of experimental autoimmune myocarditis (EAM). PROCEDURES: EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [99mTc]fucoidan single-photon emission computed tomography (SPECT)/x-ray computed tomography (CT) was performed in the very early phase of myocarditis at 10, 15, and 21 days after immunization. Then, hearts were collected and used for autoradiography, well counting, histology, and flow cytometry analysis. RESULTS: The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [99mTc]Fucoidan uptake was significantly increased in EAM compared with controls starting from D15. There was a close relationship between uptake of the tracer and myocardial content in CD45+, CD8+, CD11b+, and CD31+ cells. CONCLUSIONS: [99mTc]Fucoidan SPECT/CT accurately diagnosed the autoimmune attack in the early steps of EAM and could be used to monitor disease evolution and therapy efficiency.

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.

Core-Shell Polymer-Based Nanoparticles Deliver miR-155-5p to Endothelial Cells.

Heart failure occurs in over 30% of the worldwide population and most commonly originates from cardiovascular diseases such as myocardial infarction. microRNAs (miRNAs) target and silence specific mRNAs, thereby regulating gene expression. Because the endogenous miR-155-5p has been ascribed to vasculoprotection, loading it onto positively charged, core-shell poly(isobutylcyanoacrylate) (PIBCA)-polysaccharide nanoparticles (NPs) was attempted. NPs showed a decrease (p < 0.0001) in surface electrical charge (ζ potential), with negligible changes in size or shape when loaded with the anionic miR-155-5p. Presence of miR-155-5p in loaded NPs was further quantified. Cytocompatibility up to 100 µg/mL of NPs for 2 days with human coronary artery endothelial cells (hCAECs) was documented. NPs were able to enter hCAECs and were localized in the endoplasmic reticulum (ER). Expression of miR-155-5p was increased within the cells by 75-fold after 4 hours of incubation (p < 0.05) and was still noticeable at day 2. Differences between loaded NP-cultured cells and free miRNA, at days 1 (p < 0.05) and 2 (p < 0.001) suggest the ability of prolonged load release in physiological conditions. Expression of miR-155-5p downstream target BACH1 was decreased in the cells by 4-fold after 1 day of incubation (p < 0.05). This study is a first proof of concept that miR-155-5p can be loaded onto NPs and remain intact and biologically active in endothelial cells (ECs). These nanosystems could potentially increase an endogenous cytoprotective response and decrease damage within infarcted hearts.

Bimodal Fucoidan-Coated Zinc Oxide/Iron Oxide-Based Nanoparticles for the Imaging of Atherothrombosis.

A polyol method was used to obtain ultrasmall ZnO nanoparticles (NPs) doped with iron ions and coated with a low molecular weight fucoidan in order to perform in vivo MR and ex vivo fluorescence imaging of athrothrombosis. During the synthesis, the early elimination of water by azeotropic distillation with toluene allowed us to produce NPs which size, determined by XRD and TEM, decreased from 7 nm to 4 nm with the increase of iron/zinc ratios from 0.05 to 0.50 respectively. For the highest iron content (NP-0.50) NPs were evidenced as a mixture of nanocrystals made of wurtzite and cubic phase with a molar ratio of 2.57:1, although it was not possible to distinguish one from the other by TEM. NP-0.50 were superparamagnetic and exhibited a large emission spectrum at 470 nm when excited at 370 nm. After surface functionalization of NP-0.50 with fucoidan (fuco-0.50), the hydrodynamic size in the physiological medium was 162.0 ± 0.4 nm, with a corresponding negative zeta potential of -48.7 ± 0.4 mV, respectively. The coating was evidenced by FT-IR spectra and thermogravimetric analysis. Aqueous suspensions of fuco-0.50 revealed high transverse proton relaxivities (T2) with an r2 value of 173.5 mM-1 s-1 (300 K, 7.0 T) and remained stable for more than 3 months in water or in phosphate buffer saline without evolution of the hydrodynamic size and size distribution. No cytotoxic effect was observed on human endothelial cells up to 48 h with these NPs at a dose of 0.1 mg/mL. After injection into a rat model of atherothrombosis, MR imaging allowed the localization of diseased areas and the subsequent fluorescence imaging of thrombus on tissue slices.

Primary structure and anticoagulant activity of fucoidan from the sea cucumber Holothuria polii.

Sea cucumber fucoidan is recently endowed with a variety of biological activities. In the present study, we studied the structure and anticoagulant effect of fucoidan from the sea cucumber Holothuria polii (Fuc-Hp). The Fuc-Hp was purified by anion exchange chromatography and its structure was characterized by FT-IR and NMR spectroscopy. Molecular weight measurements were performed by HPSEC-MALLS-dRI. Fuc-Hp anticoagulant activity was measured by activated partial thromboplastin and thrombin times, and by in vitro thrombin inhibition in the presence of antithrombin and heparin cofactor II. The assessment of thrombin generation was investigated using calibrated automated thrombography. Fuc-Hp with a high sulfate content (34.6%) and an average molecular mass of 1376.3 kDa was isolated from H. polii in amount of ~2.7 mg/g dry body wall. Primary structural analysis indicated that Fuc-Hp was mainly composed of a tetrafucose repeating unit branched by glucuronic acid. Fuc-Hp exhibited a high anticoagulant effect mediated essentially by heparin cofactor II and to lesser extent by antithrombin with IC50 values of 0.16 µg/mL and 0.5 µg/mL, respectively. Overall results showed a high anticoagulant activity of Fuc-Hp, which was attributed to the high sulfate content and abundance of disulfated fucose residues of H. polii fucoidan.

Thin films of binary amorphous Zn-Zr alloys developed by magnetron co-sputtering for the production of degradable coronary stents: A preliminary study.

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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.

Characterization and anticoagulant activity of a fucosylated chondroitin sulfate with unusually procoagulant effect from sea cucumber.

The fucosylated chondroitin sulfate (FuCS) was isolated from the sea cucumber Holothuria polii body wall and then purified by anion exchange chromatography and the structure was characterized by FT-IR and NMR spectroscopy. Anticoagulant activity was measured in plasma by classical anticoagulation tests and the thrombin generation was assessed by calibrated automated thrombography. The results showed that the FuCS obtained at a yield of 4.66% possesses high sulfate content 43% and an average molecular mass of 45.8kDa. Structural analysis revealed the presence of alternating units of chondroitine sulfate branched at position 3 of the ß-d-glucuronic acid by two types of fucose 2,4-O-di and 3,4-O-disulfated residues. The FuCS exhibited a high anticoagulant effect mediated essentially by heparin cofactor II and to lesser extent by antithrombin. Moreover, the CAT assay depicted a dual effect of FuCS with a procoagulant tendency for low doses and an anticoagulant effect at higher doses.