Structure and Function of Trypsin-Loaded Fibrinolytic Liposomes.

Anna Tanka-Salamon,Miklós Lovas,Judith Mihály,Krasimir Kolev,András Wacha,Attila Bóta

doi:10.1155/2017/5130495

Abstract

Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSLT) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based SSLT were prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity of SSLT was evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C. SSLT exerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency of SSLT under dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.

Highlights

Thrombolysis based on enzymatic dissolution of fibrin is currently the first-line treatment of ischemic stroke as well as certain selected cases of acute myocardial infarction [1, 2]
Most of the fibrinolytic agents are plasminogen activators, which can be classified as “indirect fibrinolytics,” because their enzymatic action is directed towards plasminogen, while, in contrast, fibrinolytics such as plasmin and its derivatives degrade fibrin without any intermediate step of plasminogen activation and are designated as “direct fibrinolytics.”
Locally administered plasmin exerts its fibrinolytic action in fibrin-bound form being protected from its main inhibitor (α2-plasmin inhibitor) but is immediately inactivated when entering the circulation, preventing bleeding at remote sites of vascular injury

Summary

Introduction

Thrombolysis based on enzymatic dissolution of fibrin is currently the first-line treatment of ischemic stroke as well as certain selected cases of acute myocardial infarction [1, 2]. Most of the fibrinolytic agents are plasminogen activators, which can be classified as “indirect fibrinolytics,” because their enzymatic action is directed towards plasminogen, while, in contrast, fibrinolytics such as plasmin and its derivatives degrade fibrin without any intermediate step of plasminogen activation and are designated as “direct fibrinolytics.”. Locally administered plasmin exerts its fibrinolytic action in fibrin-bound form being protected from its main inhibitor (α2-plasmin inhibitor) but is immediately inactivated when entering the circulation, preventing bleeding at remote sites of vascular injury (reviewed in [4]). Drugs are preserved from metabolization prior to reaching target tissues, and simultaneously they minimize exposure of healthy tissue to the encapsulated drug during its circulation in the blood. The possibility to target liposomes helps in localizing sufficient quantities of thrombolytic agents to the desired thrombus.

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