Harnessing seaweed extract to treat ischemic stroke



World Thrombosis Day is this week and the good news is, despite pandemic restrictions, researchers have been working hard in their labs around the world to develop new innovative solutions to treat thrombosis-related conditions.


Currently for ischemic stroke, a stroke caused by a blood clot, the main treatment is an intravenous injection of recombinant tissue Plasminogen Activators (rtPA) which breaks up the clot. Studies show that patients who receive rtPA within 3-4.5hrs have better and more complete recoveries, [1]. Due to the limitations of this narrow therapeutic window and because the rate of acute recanalization (i.e. reestablishment of blood flow) is low; only 30% of patients experience full or partial recanalization [2]; it is necessary to investigate other strategies and treatment options.


On this World Thrombosis Day and in advance of World Stroke Day, taking place on 29th October, we’d like to shine a light on a paper recently published in Biomaterials that includes work from one of our customers, [3].


Study Overview

Researchers at the Université de Paris (UMR S1148, INSERM) successfully designed and fabricated fucoidan-functionalized 100% polysaccharide submicroparticles (Fuco-SPs) from biocompatible and FDA approved components as a P-selectin targeting drug delivery system for thrombolytic therapy. This mechanism is illustrated below from previous work by some of the same researchers, [4].

Graphical abstract from Maya Juenet et al. Thrombolytic therapy based on fucoidan-functionalized polymer nanoparticles targeting P-selectin, Biomaterials, Volume 156, 2018, Pages 204-216, ISSN 0142-9612, https://doi.org/10.1016/j.biomaterials.2017.11.047.

And if you’re wondering what seaweed has to do with this?

Fucoidan is a long chain sulfated polysaccharide found in various species of brown algae and commercially available fucoidan is commonly extracted from the seaweed species.


In vitro dynamic flow assay with Cellix’s Vena8 Fluoro+ biochips & ExiGo microfluidic pump

To date, most published articles focus on in vitro static assays where such targeted therapies are assessed via flow cytometry or confocal microscopy. In this work, researchers established an in vitro dynamic flow microchamber assay with Cellix’s ExiGo pump and Vena8 Fluoro+ biochips that mimicked arterial or venous blood flow conditions with the aim of studying targeting efficacy for recombinant P-selectin and/or human activated platelet aggregates expressing P-selectin.


Microchannels of the Vena8 Fluoro+ biochips were pre-coated with P-selectin and using the ExiGo pump, fluorescent Fuco-SPs and Control-SPs were subsequently injected into the microchannels at arterial or venous shear rates (67.50 dyne/cm2 vs. 6.75 dyne/cm2) and their adhesion was visualised and quantified, see Figures below. Fuco-SPs specifically adhered to the recombinant P-selectin in a dose-dependent manner, but not to E- and L-Selectins.

Zenych et al., Fig. 3 (A, E) Evaluation of the SPs interactions with selectins. A. Adhesion of the Control-SPs or Fuco-SPs on the coating of the recombinant P-selectin in the microfluidic assay under arterial and venous flow conditions (n = 4). E. Fluorescent microscope view of Control-SPs or Fuco-SPs adhesion over a P-selectin coating of 100 μg/ml under arterial and venous shear rates (scale bar = 20 μm), [3].

Before moving onto a murine model, researchers continued with the microfluidic experiments and validated the capability of the Fuco-SPs to actively anchor onto the surface of activated platelets, expressing P-selectin. The Vena8 Fluoro+ biochips were pre-coated with collagen to induce platelet activation and aggregation and the ExiGo pump was used to inject human whole blood.

Zenych et al. [3], Fig. 5 (A, B) Adhesion of the SPs over activated platelet aggregates. A. Visualization by fluorescent microscopy of the attached unloaded SPs on the microchannels after the formation of the platelet aggregates (scale bar = 20 μm). B. Corresponding quantification of the integral density of the unloaded (n = 6) and rtPA-loaded (n = 4) Control-SPs and Fuco-SPs in ImageJ.

Fluorescent Fuco-SPs or Control-SPS were subsequently perfused at arterial shear tress and the Fuco-SPs were clearly detected with significantly more adhesion on the surface of the activated platelet aggregates when compared to the Control-SPs (Fig. 5A). It is important to note that adsorption of rtPA did not impair the Fuco-SPs clot-binding ability (Fig. 5B).

Researchers also developed a murine model of ischemic stroke that rtPA conjugation to the Fuco-SPs could enhance the thrombolytic activity of the clinical agent in vivo. The blood flow perfusion was restored more rapidly, which resulted in smaller post-ischemic cerebral infarct lesions and higher BBB protection.


Summary

In summary, a rtPA-associated Fuco-SPs present an exciting alternative to existing therapies which may in the future provide patients with an improved thrombolytic therapy in terms of safety and efficacy.

Planning a similar experimental study?

Consider the advantages of the Vena8 Fluoro+ Biochip



Cellix biochips simulate human capillaries to aid the accurate and the Vena8 Fluoro+ biochip is ideal for platelet adhesion and aggregation shear flow experiments, particularly at high shear rates. The chip is robust, simple, and easy to use.




Here's how it works:

The microchannel (which simulates a blood vessel) is coated with an adhesion molecule suchas vWF, collagen, fibrinogen, etc. The biochip is then placed on a microscope frame and connected to the Mirus Evo pump.

The prepared cell sample (whole blood or purified platelets) is then pumped through the microchannel at the user’s defined shear rate, which may be ramped up or down. The platelets engage with receptors on the microchannel wall resulting in adhesion, aggregation and thrombi formation.


Biochip Details:

  • Microchannel Dimensions: 400µm (width) x 100µm (depth) x 20mm (length).

  • 8 channels per biochip = 8 assays with lots of different possibilities - for example, you can coat each channel with a different adhesion molecule.

Features and Benefits of Vena8 Fluoro+

  • Compatible with confocal microscopy and fluorescence immunostaining.

  • Plug and play - Compact with easy to connect tubing.

  • Thrombosis, platelet adhesion, and aggregation flow-based assays with shear rate range from 50 - 10,000 s−1 with ExiGo or Mirus Evo pumps.

  • Reagent savings - only 10µL ligand/protein coating per channel (e.g. VWF, collagen, etc.) with a standard pipette.

  • Low sample volume even at high shear rates with whole blood or cell suspensions.

Click here to find out more information.


References

1. W.J. Powers et al. Guidelines for the early management of patients with acute ischemic stroke: 2019 update to the 2018 guidelines for the early management of acute ischemic stroke: a guideline for healthcare professionals from the American heart association/American stroke, Stroke 50 (2019) e344, https://doi.org/10.1161/STR.0000000000000211. –e418.

2. R. Bhatia et al. Low rates of acute recanalization with intravenous recombinant tissue plasminogen activator in ischemic stroke: real-world experience and a call for action, Stroke 41 (2010) 2254–2258, https://doi.org/10.1161/ STROKEAHA.110.592535.

3. Alina Zenych et al. Fucoidan-functionalized polysaccharide submicroparticles loaded with alteplase for efficient targeted thrombolytic therapy, Biomaterials, Volume 277, 2021, 121102, ISSN 0142-9612, https://doi.org/10.1016/j.biomaterials.2021.121102.

4. Maya Juenet et al. Thrombolytic therapy based on fucoidan-functionalized polymer nanoparticles targeting P-selectin, Biomaterials, Volume 156, 2018, Pages 204-216, ISSN 0142-9612, https://doi.org/10.1016/j.biomaterials.2017.11.047





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