In vivo behaviour of long‐circulating liposomes in blood vessels in hamster inflammation and septic shock models—use of intravital fluorescence microscopy

Abstract

This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5 and 6-CF-encapsulated polyethylene glycolated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope (using x40, x80 magnification) fitted with a Xenon light source and an epifluorescence assembly (excitation, 470 nm, FWHM 40 nm; emission, 540 nm, FWHM 40 nm). An ultra-high sensitivity videocamera mounted on the microscope projected the image onto a monitor, and the images (720 x 576 pixels) were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using this model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 h. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up, liposomes. This observation is in accordance with previous in vitro studies.

Revealing Dynamics of Accumulation of Systemically Injected Liposomes in the Skin by Intravital Microscopy

“…Images of liposomes in PBS or mouse serum in vitro and images of plasma and blood cells recovered in vivo 15 min postinjection showed the absence of aggregates and no visible uptake by blood cells (Figure 2E and Figure S3). Moreover, in contrast to a previous report on the uptake of PEGylated liposomes by platelets after injection into mice, 22 we did not observe fluorescent platelets in platelet-rich plasma (Figure S3). In order to address the concern that the fluorescent label dissociated from liposomes soon after the injection, non-PEGylated or PEGylated EPC liposomes were incubated in mouse serum for 1 h and analyzed for the presence of the dye in serum supernatant (after pelleting the liposomes by ultracentrifugation).…”

Liposomes and Blood Cells: A Flow Cytometric Study

“…As well, a transfer of platelet membrane proteins to the liposomes (Dalencon et al, 1996;Okumura et al, 1994) can be accomplished by a variety of liposome formulations. Red cells are also able to do this (Sato et al, 1990;Suzuki and Okumura, 2000) as can leukocytes (Devoisselle et al, 2001;Gerber et al, 2001). In many cases platelet-liposome fusion is not detrimental to platelets (Male et al, 1992): but may (Juliano et al, 1983) or may not cause (Nishiya and Sloan, 1996;Stensrud et al, 1999) overt changes of platelet function such as aggregation or hemostasis.…”

<title>Intravital fluorescence microscopic study of the behavior of long-circulating liposomes during microvascular thrombosis</title>

“…Some advantages of liposomes staining by carboxyfluorescein have been already described (6). 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.…”

“…size, charge). In a previous study, we have already demonstrated that long-circulating liposomes are able to interact with leukocytes in a inflammation site using fluorescent labeled liposomes and fluorescence intravital microscopy (6). PEGylated liposomes are taken up by leukocytes in flowing blood.…”