Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with Positive Contrast in Magnetic Resonance Imaging for Specific Biomedical Applications.

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH) are well-known for their anticoagulant properties. There is also currently a growing interest in using LMWH in targeted cancer therapy. In particular, several types inhibit heparanase, a key enzyme overexpressed in the tumor microenvironment that promotes angiogenesis progression and metastasis spreading. Here, we propose iron oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging. As a proof of concept, magnetic resonance angiography (MRA) was conducted in mice up to 3 h after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enable innovative theranostic applications if combined with the various bioactivities of the heparins. Indeed, we showed, using advanced in vitro tests, how HEP-IONP anticoagulant or anti-heparanase activities were maintained depending on the heparin species used for the coating. Overall, the study allowed presenting an IONP coated with a commercial LMWH (Lovenox) suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Production of heparin and λ-carrageenan anti-heparanase derivatives using a combination of physicochemical depolymerization and glycol splitting.

Strongly associated with tumor angiogenesis and metastasis, the enzyme heparanase is an endo-ß-d-glucuronidase which is overexpressed in the tumor microenvironment. Its inhibition could be one of the most promising anti-angiogenic approaches to date. Although heparin is known as a good heparanase inhibitor, it also possesses major anticoagulant properties that may be incompatible with its use as an anti-angiogenic agent, hence the considerable interest for other sources of sulfated polysaccharides. Recent investigations point to λ-carrageenans, highly sulfated galactans with a tremendous potential that are found in red algae. This study describes the production of low-molecular-weight (LMW) heparins and λ-carrageenans, using a combination of glycol splitting and ultrasonically-assisted radical hydrolysis using hydrogen-peroxide. The structural characteristics, as well as the anticoagulant and antiheparanase activities of the resulting products were assessed. The best candidate was a LMW glycol-split λ-carrageenan that displayed major anti-heparanase properties, with an IC50 of 7.32ng/mL and a close-to-zero anticoagulant activity.

Anti-heparanase activity of ultra-low-molecular-weight heparin produced by physicochemical depolymerization.

Heparanase is an endo-ß-D-glucuronidase that plays an important role in cancer progression, in particular during tumor angiogenesis and metastasis. Inhibiting this enzyme is considered as one of the most promising approaches in cancer therapy. Heparin is a complex glycoaminoglycan known as a strong inhibitor of heparanase. It is primarily used in clinical practice for its anticoagulant activities, which may not be compatible with its use as anti-angiogenic agent. In this study, we described the production of ultra-low-molecular-weight heparins (ULMWH) by a physicochemical method that consists in a hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. We assessed the structural characteristics, anticoagulant and anti-heparanase activities of the obtained heparin derivatives and compared them with three commercial low-molecular-weight heparins (LMWH), glycol-split non-anticoagulant heparins and heparins produced by enzymatic methods. ULMWH generated by the physicochemical method were characterized by high anti-heparanase and moderate anticoagulant activities. These heparin derivatives might be potential candidates for cancer therapy when a compromise is needed between anti-heparanase and anticoagulant activities.

Alteration of cathepsin D trafficking induced by hypoxia and extracellular acidification in MCF-7 breast cancer cells.

The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.