טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLevi Moran
SubjectDrug Carriers Functionalized with GPVI for Target Drug
Delivery
DepartmentDepartment of Biomedical Engineering
Supervisor Assistant Professor Netanel Korin
Full Thesis textFull thesis text - English Version


Abstract

therothrombosis, characterized by atherosclerotic plaque disruption with superimposed thrombosis, is a major cause of acute coronary syndromes (ACS) and a leading cause of mortality in the Western world.  At sites of atherosclerosis plaque disruption, thrombosis occurs through the interaction of platelets, clotting factors, subendothelial proteins, and atheromatous debris, exposed on atherosclerotic lesion. Platelets play a key role in this process by adhering to collagen directly through Glycoprotein VI (GPVI) and indirectly through von Willebrand factor (vWF) and glycoprotein Ib (GPIb) interaction. Arterial thrombi are mainly composed of aggregated platelets and antiplatelet medication is the current strategy to deal with thrombosis and inhibit platelet aggregation. However, there are also bleeding risks that need to be considered. Inspired by circulating platelets’ innate ability to target vascular injuries, nanoparticles functionalized with platelet receptors can be optimized as drug carriers designed to selectively target sites of thrombosis. GPVI is a platelet specific collagen receptor, when it takes its dimeric form it shows high affinity to collagen. The dimeric GPVI has been shown to inhibit thrombus formation in preclinical studies and in a phase I clinical trial with minimal effect on hemostasis.   However, so far GPVI has not be utilized as at targeting moiety for targeted drug delivery and targeted nanomedicine to sites of atherthrombosis.

In this work, the adhesion kinetics of nanoparticles functionalized with GPVI under physiological flow conditions is studied. First, fluorescently labelled Poly (lactic-co-glycolic acid) (PLGA) nanoparticles were fabricated, in two sizes (200 nm and 2 μm) via the oil-in- water emulsion-solvent evaporation method, and then surface functionalized with GPVI through EDC-Sulfo NHS chemistry. The particle solution was then perfused in collagen coated microfluidic channels and their adhesion under flow was monitored using confocal time-lapse microscopy.

The results showed enhanced, specific adhesion of both the 2 μm and 200 nm GPVI functionalized particles compared to control, BSA functionalized particles. Adhesion kinetics were shown to be shear dependent within physiological levels of shear and were highly influenced by particle size. We observed that the 2 μm GPVI coated particles did not adhere to collagen above 10 dyne/cm2 while, the 200 nm GPVI coated particles adhered throughout all shear stress conditions (1-40 dyne/cm2). Additionally, unlike the GPVI particles, the BSA coated particles showed low adhesion at low shear and no adhesion at high shear.  From these results we calculated the relative affinity to collagen of the GPVI coated particles comparing to the BSA coated particles and we saw that the relative affinity to collagen increases with shear for the 200 nm particles. We also assessed the adhesion probability as function of shear stress and particle size of the GPVI coated particles and showed that it decreases with shear stress for the 200 nm particles. Finally, preliminary results in real-sized, coronary artery mimicking stenosis models coated with collagen showed a shear dependent spatial adhesion pattern of the 2 μm GPVI coated particles. The particles preferably adhered to low shear areas mainly at the post- stenotic region.

 Altogether, this study highlights the need for applying engineering approaches and tools when designing and optimizing targeted cardiovascular nanomedicines.