Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic Lipoplexes Earlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (1). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled. The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60% of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90% of the particles was in the blood. Currently, we compare the encapsulation efficiency, stability and targetability of the CCL with stabilized antisense lipid particles (SALP) (2), while also the biological activity of these carriers is addressed. In conclusion our results demonstrate that antisense ODN can be targeted very efficiently to EC in vivo, employing plasma-stable CCL, surface modified with negatively charged albumin. ReferencesStuart DD, Allen TM. BBA 2000; 1463:219-229.Semple S. et al. BBA 2001; 1510: 152-166. Localisation of Deep Vein Thrombosis Using Drug Delivery System Containing Radioactive Streptokinase The use of radionuclides as tracers for localisation and identification of deep vein thrombosis has been a subject of much interest in the past several years. A number of research have been performed for localisation of deep vein thrombosis (DVT). Unfortunately, none of these developed agents was used in clinic because of some disadvantages. For this reason, experiments are still going on to develop better radiopharmaceuticals for scintigraphic imaging of DVT. Streptokinase produced by Lancefield Group C β-hemolitic streptococci has been found to induce dissolution of already formed venous and arterial thrombi. Because, the therapeutic use of streptokinase has been reported in the treatment of venous and arterial thrombosis, usefulness of radiolabelled streptokinase a possible agent for localisation of an already formed thrombus has been investigated. But, streptokinase is rapidly removed from circulation so enough amount of streptokinase does not accumulate in thrombi. In this study, streptokinase was entrapped into liposome, niosome and sphingosome dispersions to improve the clot selectivity. Liposome, niosome, sphingosome dispersions containing streptokinase were prepared by film method followed by extrusion and freeze-thawing. 80-91% of activity of enzymes was protected after the preparation when compared with the initial activity. Entrapped drug amount and vesicle size were determined as 10-13% and 150-200 nm, respectively. These systems were tested in an in vivo rabbit model by forming the clots in the jugular vein by injection of thrombin. For biodistribution studies, firstly, RES organs were saturated by injection of empty dispersions to avoid of high uptake of vesicles from RES especially liver. After the injection of free streptokinase and dispersions containing streptokinase, animals were sacrified and RES organs, kidney, lung, vein and thrombus were removed and washed with saline. Then radioactivity of each organs were counted by gamma counter and uptake % per gram organ was calculated for each organ. Important parameter for the biodistribution results was the comparison of the Thrombus Vein ratio. With the entrapment of the streptokinase in the vesicles, thrombus uptake and imaging quality were improved and a high Thrombus Vein ratio was obtained (p<0.05). For scintigraphic imaging studies, after the saturation of RES with empty dispersions, liposome niosome sphingosome dispersions containing streptokinase were injected to the animals. Scintigraphic scans were obtained every 15 m up to 90 m. postadministration. Positive scans indicating uptake of radioactivity were noted as early as 15 m after administration of liposomes containing 99mTc-streptokinase and images quality improved in time. It can be seen from the results of biodistribution studies, liposome niosome sphingosome dispersions containing streptokinase are giving the promising results for the future research. Certainly, further studies are required to improve the quality of scintigraphic images (1). References Erdo an S. In vitro and in vivo studies on drug delivery system developed for diagnosis and scintigraphic imaging of deep vein thrombosis, Ph.D. Thesis, Hacettepe University, Ankara, 2001. Formation of Bilayer Discs in Lysolipid-Containing Thermosensitive Liposomes During Phase Transition: A New Drug Release Mechanism Lysolipid-containing thermosensitive liposomes (LTSL) containing the anticancer drug doxorubicin (DOX) exhibit superior efficacy in a human tumor xenograft mouse model over lysolipid-free thermosensitive liposomes (TSL) and non-thermosensitive liposomes (NTSL). The reason for this superior efficacy is the increased local drug bioavailability after applying mild hyperthermia to the tumor site resulting in almost instant drug release from LTSL. In the present study, we investigate the drug release mechanism and provide evidence that incorporation of lysolipid in the phospholipid membrane favors the formation of bilayer discs at the phase transition temperature (TC). LTSL and TSL were prepared and kept above TC or cycled trough TC before extrusion. Cryogenic transmission electron microscopy images of samples vitrified above and below TC revealed that bilayer discs were initially not present in the preparation but formed as liposomes were cycled through TC. The amount of bilayer discs increased with the number of TC cycles and was dependent on the presence of lysolipid in the liposomal membrane. The DOX loading capacity decreased with an increasing number of TC cycles and more so in LTSL than in TSL but not in NTSL. We hypothesize that at TC, lysolipids can segregate in phase boundaries between gel-phase plates and adapt a micellar confirmation within the phospholipid bilayer at the rim of bilayer plates. Bilayer discs can then dissociate from liposomes leading to an instant release of the liposomal content. Thus, disc formation may be regarded as a new drug-release mechanism in lysolipid-containing cholesterol-free thermosensitive liposomes. Liposomal Therapeutics: Process and Formulation Aspects The literature of liposome science is dominated by the issue of formulation (e.g. lipid composition) and by gross morphological status (e.g. MLV vs. SUV). Relatively less emphasis is placed on production process, process optimization, quality control of raw materials, or in-process and finished product quality control testing. Liposomal preparations of identical composition can exhibit very different performance characteristics depending on process conditions. We will present examples of such process and control issues from Gilead's commercial products AmBisome® (liposomal amphotericin B) and DaunoXome® (liposomal daunorubicin), and from the investigational products MiKasome (liposomal amikacin), NX211 (liposomal lurtotecan), GS7904L (liposomal GW1843U89), and pre-clinical stage liposomal products. Accelerated Blood Clearance of TRX-Liposomes in Rats and Cynomolgus Monkeys Accelerated blood clearance of long-circulating liposomes was first reported by Dams et al. (JPET 292:1071-1079, 2000), however, the details of this phenomenon are still unknown. We have developed a novel PEG-liposomal formulation coated with novel cationic lipid (TRX-20). TRX-liposomes have long-circulating characteristics and can target tissues expressing specific types of glycosaminoglycans. In this study, we investigated whether TRX-liposomes show the accelerated blood clearance upon repeated injection. Furthermore, the phenomenon was evaluated in Sprague-Dawley rats and Cynomolgus monkeys to investigate the effect of the species. TRX-liposomes were injected once or twice a week in rats and every two weeks in Cynomolgus monkeys during eight weeks. In rats, the blood concentration of the liposomes one week after first injection were decreased significantly in the both case of injection once a week and twice a week, indicating that the TRX-liposomes showed the phenomenon. The phenomenon, however, attenuated thereafter and disappeared after eight weeks. The phenomenon was independent of injection and total injection of lipid dose. On the other hand, in Cynomolgus monkeys, the plasma concentration profiles of liposomes didn't change during two months. These results suggest that the accelerated blood clearance phenomenon is highly species-specific, probably due to the differences in the immune system between the species. Uptake and Intracellular Processing of PEG-Liposomes and PEG-Immunoliposomes by Kupffer Cells In Vitro Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) (PEG) chains on the surface of the immunoliposomes may, however, attenuate this effect. We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with 3H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular proc