Nanovesicles dissolve blood clots reducing side-effects

Blood clots, or thrombi, are comprised of aggregated platelets and red blood cells with a mesh of cross-linked fibrin protein that can obstruct or block the normal flow of blood in blood vessels. This can lead to strokes or heart attacks. Treatments available include a clot-dissolving drug called tissue plasminogen activator (tPA). However, this therapy has limitations such as a short circulation time; and side effects such as excessive bleeding.

But now, researchers at Imperial College London have devised a clever solution to encapsulate and protect tPA in the blood stream in the form of a nanovesicle. This tPA-loaded nanovesicle enables tPA to specifically bind to thrombi under flow conditions and to efficiently trigger the release of tPA locally in a controlled manner.

Dr. Rongjun Chen of Imperial’s Department of Chemical Engineering said:

“tPA has a narrow window between the desired effect and side effects, so we have wrapped it in a package that extends this therapeutic window and minimizes the required dose. Our results are exciting, but animal and clinical studies are required for validation.”

Study Overview

How does the nanovesicle work?

Huang et al., Fig. 1B: Schematic illustration of a fibrinogen-mimicking, multiarm lipid nanovesicle, denoted as tPA-cRGD-PEG-NV
tPA encapsulated nanovesicle

Blood clots are formed when activated platelets bind together with fibrinogen proteins which cause a “bridging effect” inducing platelet aggregation. By mimicking fibrinogen on the surface of the nanovesicle, the researchers (Huang et al.) were able to create targeted nanovesicle encapsulating tPA which selectively binds to activated platelets and importantly triggers release of tPA selectively at the site of a blood clot.

Since platelets become activated under shear stress, it was important to test the new nanovesicles under flow conditions. The researchers at Imperial College London used Cellix’s VenaFlux solutions to simulated blood vessels, an example of their set-up is shown below.

Thrombus selectivity under physiological flow conditions

First, the researchers demonstrated that the nanovesicles, did in fact, selectively target the blood clots under physiological flow conditions. This was done by:

  • Creating blood clots: first, citrated human blood was perfused in the collagen-coated Vena8 Fluoro+ biochip.

  • Label the nanovesicles: nanovesicles were labelled or stained with fluorescein isothiocyanate (FITC).

  • Perfuse the FITC-labelled nanovesicles in the biochip containing the blood clots: images and data analysis illustrated a statistically significant difference compared to controls as shown in the images below. This clearly demonstrated the specific targeting of the nanovesicles to blood clots under physiological flow conditions.

Huang et al., Fig. 3C and 3D: (C) Representative bright-field and fluorescence images of human blood clots in the flow chamber after perfusion with the FITC-labeled tPA-PEG-NV and tPA-cRGD-PEG-NV, respectively. (D) FITC fluorescence intensity in the fluorescence images of human thrombi after incubation with the FITC labeled tPA-PEG-NV and tPA-cRGD-PEG-NV, respectively. Data are presented as the average   SD (n ≥ 3). Statistical analysis was performed using the Student’s t test. *P < 0.05. Photo credit: Yu Huang, Imperial College London.
FITC-labelled tPA encapsulated nanovesicle (tPA-cRGD-PEG-NV) selectively binds to blood clots under physiological flow conditions

Targeted thrombolysis under flow conditions

Researchers also investigated the ability of these tPA-encapsulated nanovesicles to efficiently dissolve the clots under similar physiological flow conditions – and compared this to traditional methods of free tPA treatment (i.e. tPA that was not encapsulated in a nanovesicle).

Huang et al., Fig. 7C, 7D and 7E: (C) tPA-encapsulated nanovesicles (tPA-cRGD-PEG-NV) perfused in microchannels with pre-stained blood clots (platelet green, fibrin red). (D) Real-time changes in the red fluorescence of fibrin at human thrombi after perfusion with cRGD-PEG-NV, tPA-PEG-NV, and tPA-cRGD-PEG-NV, respectively.  (E) Time required for complete human blood clot removal after perfusion with control (tPA-PEG-NV) and tPA-encapsulated nanovesicles (tPA-cRGD-PEG-NV) and free tPA.
Blood clots (platelets green, fibrin red) in Vena8 Fluoro+ biochips treated with control and tPA-encapsulated nanovesicles (tPA-cRGD-PEG-NV) under physiological flow conditions

Again, the researchers used Cellix’s VenaFlux solutions to mimic human blood vessels:

  • Creating (stained) blood clots: first, recalcified citrated blood labelled with DIOC6 (staining platelets green) and AF-647-FBG (staining fibrin red) was perfused in the collagen- and tissue factor (TF)-coated channels of the Vena8 Fluoro+ biochip.

  • Perfuse control and tPA-encapsulated nanovesicles into the biochip: nanovesicles were added to recalcified blood and perfused into the biochip containing the blood clots.

  • Dissolving the blood clot: Dissolution of blood clots were monitored in real-time and quantified as a decrease in fluorescence intensity of platelets and fibrin.

Treatment of blood clots with tPA-encapsulated nanovesicles resulted in fluorescence of fibrin and platelets completely disappearing and facilitated complete clot removal. The time required was comparable with free tPA.

Co-author Professor Simon Thom of Imperial’s National Heart and Lung Institute said:

“We’ve found a way to make a clot-busting drug more precisely targeted, potentially enhancing efficacy and reducing catastrophic side effects. This good work paves the way for safer delivery of drugs with otherwise harmful side effects and demonstrates the activity of nano-encapsulated tPA in a laboratory setting. Research is now needed in whole organisms to determine the capsule’s effectiveness in a more realistic setting.”

What do I need to get started?

Interested in setting up a similar experiment in your lab but not sure where to start?

Here’s what you’ll need:

  • Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below.

  • Mirus Evo pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels.

  • Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

  • Image Pro Cell Analysis software – to analyse the images and videos from your experiments.

You may already have some of these items in your lab (e.g. such as an inverted microscope, camera and cell analysis software). In this case, we would recommend the VenaFlux Starter kit. We have options to suit all budgets so contact us now to learn more about our products or request a quote.

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