Behavior of platelets stained by 5,6-CF-encapsulated PEGylated liposomes after laser irradiation of vessel wall: an in-vivo model for studying site-selective delivery of diagnostic or therapeutic agents

Abstract

Vascular endothelium serves as an extensive interface between circulating blood and various tissues and organs of the body. As such, it offers an accessible target for blood-borne pharmacological and genetic manipulations that can mediate both local and systemic effects. Thus, targeting of liposomes to activated vascular endothelial cells may provide a strategy for site-selective delivery in the vascular system with broad therapeutic applicability. This study aimed to evaluate an intravital fluorescence imaging technique to visualize in-situ and in real-time the activation of platelets after staining by 5,6-CF- encapsulated PEGylated liposomes injected intravenously. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. The skin micro circulation was observed with an intravital microscope (using x25 and x40 magnification) fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for play-back analysis with a digital video cassette recorder. An inflammatory response was induced by an Argon laser emitting at 514.5nm. The 80micrometers laser beam was focused on a vessel and its position was controlled with the microscope imaging system, it was possible to see individual platelets flowing in blood vessels. As liposomes were labeled with a fluorescent probe which was hydrophilic (located in the aqueous phase), the fluorescence of platelets was due only to the uptake of liposomes. After laser irradiation, platelets activation at sites of vascular injury was obtained. Tethering, translocation of some platelets inside the irradiated zone were clearly seen. At last, detachment and extravasation of platelets were observed. A perivascular fluorescence confirmed that platelets migrated across the basal lamina into the dermal connective tissue. In conclusion, staining of platelets using 5,6-CF-encapsulated PEGylated liposomes injected intravenously presents the following advantages: i) in-situ labeling, ii) use of hydrophilic marker located in an aqueous compartment within the platelet, iii) as the release of the fluorescence marker is slow due to the formulation of liposomes, labeling of platelets could be observed during the whole experiment. Laser irradiation of blood vessels in vivo can induce the different phases of platelet activation: i) recruitment, ii) adhesion, iii) detachment, iv) transmigration. The combination of these techniques (platelet staining with PEGylated liposomes, intravital fluorescence microscopy, laser irradiation) provides a powerful tool to study local inflammation, platelet activation and behavior of liposomes in situ and in real time at an inflammation site. These observations could be considered as a preliminary approach to study the targeting of drugs to an endothelium under inflammation environment.