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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) (). 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) (), 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. Stuart DD, Allen TM. BBA 2000; 1463:219–229. Semple S. et al. BBA 2001; 1510: 152–166. 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 (). Erdoğan 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. 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. 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 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. 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 processing of the liposomes by the Kupffer cells, based on a double-label assay using the 3H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-14C-oleate as relative measure of degradation. Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes. In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface. These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity. Koning GA, Morselt HWM, Kamps JAAM, Scherphof GL. J Liposome Research 2001; 11:195-209 Although rheumatoid arthritis is a chronic, progressive autoimmune disorder, exacerbation and quieter periods often characterize the development of joint inflammation. As compared to the quieter stages of the disease, exacerbation is generally hard to control. Often glucocorticoids are necessary for successful intervention. Serious adverse effects limit however prolonged systemic use of glucocorticoids while local intra-articular injections are restricted to single joints only. Here we present a targeting approach to enhance local delivery of glucocorticoids at arthritic joints. We encapsulated prednisolone in small long-circulating liposomes, which extravasate in inflamed tissue after i.v. administration. Liposomal prednisolone proved to be highly effective in different arthritis models: rat adjuvant arthritis as well as collagen arthritis. A single injection resulted in complete reversal of the entire inflammation response for almost a week. In contrast, the same dose unencapsulated prednisolone had a limited effect after repeated daily injections only. We collected further evidence that targeted steroid delivery in the inflamed joint is the key factor explaining the observed therapeutic benefit, excluding other possible explanations like splenic accumulation or prolonged release of prednisolone in the circulation. Evaluating and comparing different glucocorticoids in long-circulating liposomes revealed that encapsulation of topical glucocorticoids with high local vs. low systemic activity can even further increase the benefit-risk ratio. Thus, targeted delivery using long-circulating liposomes may provide a novel approach to successful glucocorticoid therapy of rheumatoid arthritis. This project is part of UNYPHAR, a research network between Yamanouchi Europe BV and the Universities of Groningen, Leiden and Utrecht Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood. Since intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor, novel therapeutic approaches are needed. Immunoliposomes (SIL) have been previously shown to enhance the selective localization of their entrapped drugs to solid tumors, with improvements in therapeutic indices. NB tumor, but not normal tissues, over-express the internalizing epitope disialoganglioside (GD2) at the cell surface. In this study we report the selective cytotoxic effect of doxorubicin (DXR) on several GD2-positive NB cell lines when the drug is administered in anti-GD2-targeted immunoliposomes (aGD2-SIL[DXR]) in vitro. Pharmacokinetic studies showed their long-circulating profiles in blood. In an in vivo NB metastatic model, our results show that aGD2-SIL[DXR] inhibited the development of macroscopic and microscopic metastases in several organs (mainly adrenal gland, kidneys and bone marrow) compared to animals treated with free-aGD2 mAb, free-DXR or DXR encapsulated in non-targeted liposomes (SL[DXR]). Further, aGD2-SIL[DXR] were more effective at prolonging survival time than SL[DXR] or free drug with all animals treated with aGD2-SIL[DXR] still alive after five months. Mice treated with free-aGD2 mAb showed partial, but significant, increases in life span. In another set of experiments anti-GD2 Fab'-fragments were used to target immunoliposomes with entrapped DXR (Fab'-SIL[DXR]). This new formulation maintained specific binding, uptake and cytotoxic effects on several GD2-positive NB cell lines. Preliminary data on the therapeutic efficacy of Fab'SIL[DXR] show increased life spans and prolonged survival times only in mice treated with Fab'-SIL[DXR] compared to animals treated with free anti-GD2 Fab' fragments, free-DXR or SL[DXR]. Further experiments will provide the ultimate determination of the utility of Fab'-SIL[DXR] formulations for the treatment of neuroblastoma in minimal residual disease or advanced disease stages. Supported by Fondazione Italiana per la lotta al Neuroblastoma and Costa Crociere. The usefulness of vaccinating fish by intramuscular injection of naked DNA encoding proteins from pathogenic fish viruses has been well documented. Less focus has been on the most common way of administering vaccines to fish, intraperitoneal injection. Therefore, the aim of this study was to assess expression in internal organs of a gene encoding luciferase after intraperitoneal injection. DNA was injected intraperitoneally as naked, formulated as lipoplex 2 (DOPE:DOTAP 1:1) or as lipoplex 5 (DOPE:DOTAP 1:1 or DOPE:DC-CHOL 1:1) to rainbow trout fry. Naked DNA was injected intramuscularly as a positive control. Organs (spleen, liver, head kidney) and muscle at the injection site were assessed for luciferase activity four days after the delivery of DNA. Differences were found between non-formulated and formulated DNA as well asbetween the different lipoplex formulations. The highest levels of luciferase activity in the liver were found after intraperitoneal injection of lipoplex 2. Also in the head kidney, the lipoplex 2 formulation gave highest levels of luciferase activity. Spleen showed highest levels after injection of naked DNA, but only marginally higher than lipoplex 2. Intraperitonal administration of DNA vaccines may be a promising route of delivery, conditional on liposome formulation being used. The ortho-carboranyl lactoside, [1,2-dicarba-closo-dodecarboran (12)-1-ylmethyl](β-d-galactopyranosyl)-(1→4)-β-gluco-pyranoside, FB22, is a relatively new "third generation" compound to be used in Boron Neutron Capture Therapy (BNCT). Liposome formulations containing FB22 were prepared as potential agents to deliver boron compounds in tumor cells. In particular liposomes composed of EPC, EPC/Cho and DOTAP/DOPE (molar ratio 1:1) were loaded with the carboranyl-lactoside and the resulting dispersions were characterized by Electron Spin Resonance spectroscopy (ESR) and cryo-Transmission Electron Microscopy (cryo-TEM). FB22 is an amphiphilic carbohydrate-carborane hybrids consisting of a lipophilic core (carborane cage) and a glycoside moiety for conferring high affinity recognition by the cellular lectines. ESR of membrane-soluble nitroxides (doxylstearic acids, n-DSA, located in different regions of the liposome bilayer) clearly demonstrated the insertion of FB22 in the liposome membrane with resultant appreciable modification of the dynamic properties of the bilayer. The analysis of the computed ESR line shape of n-DSA showed an increase of order parameter and mobility of the liposome bilayer where the FB22 was localized (S20 from 0.5 to 0.63 and τc from 3 × 10−10 to 1.1 × 10−9 on going from 0 to 0.66 mol% of FB22). Cryo-TEM micrographs strongly suggested that the carboranyl-lactosides were embedded within the liposome bilayer. We observed the formation of large liposomes clusters at relatively low FB22 to lipid ratios and at these values the liposome bilayer integrity was preserved. The progressive increase of the lactosyl-carborane produced a dramatically change of the aggregate shape. Some liposomes showed stabilized openings in the bilayer structure and threadlike micelles, which were originated from the liposome membrane as complex networks, came out in the external matrix or were encapsulated in the liposomal aqueous core. New blood vessel formation, angiogenesis, is an essential process in the development of a clinically relevant tumour. Inhibition of the formation of new capillaries or blocking the angiogenic vessels would be an interesting strategy in tumour therapy. One of the most attractive target cell types in this approach are the endothelial cells (EC), as they play a pivotal role in the angiogenic cascade. One of the proteins overexpressed on angiogenic EC, for which a targeting ligand exists, is the αvβ3-integrin. Synthetic cyclic RGD-peptides show a high affinity for this integrin. By coupling of RGD-peptides to long-circulating liposomes, the targeting potential of the peptides may be combined to the favourable pharmacokinetic profile and drug transport capacity of liposomes. Incubation of RGD-peptide targeted liposomes (RGD-L) with HUVEC resulted in 7-fold higher binding than for non-targeted liposomes (L) and liposomes bearing the control RAD-peptide (RAD-L). By CLSM it was determined that RGD-L are, after binding to the cells, taken up inside endocytic vesicles. For RAD-L and L hardly any bound or intracellular fluorescence could be detected. In vivo studies in tumour-bearing mice show that the circulation time of RGD-L is reduced as compared to RAD-L and L, the latter two formulations showing similar circulation kinetics. Interestingly, despite the increased clearance rate of the RGD-L, localization in the tumour was similar. As the main driving force for tumour localization of liposomes is regarded to be the liposomal AUC in the circulation, RGD-L localization was expected to be lower. These findings indicate a specific interaction at the site of the tumour. Using intravital microscopy, the specific interaction of RGD-L with the blood vessel wall at the target site could indeed be visualized. Transfersomes®, a trademark of IDEA-AG, are specially designed drug carriers for non-invasive delivery across the skin. Their very high shape adaptability, controlled mainly by formulation parameters, and their spontaneous ability to cross the skin, controlled by administration conditions, ensure efficient transport of such carriers and associated pharmaceuticals through the skin. Superoxide radicals are involved in a variety of pathologic situations, such as inflammation. Superoxide dismutase (SOD), with antioxidant and anti-inflammatory properties, has been suggested as a therapeutic agent for treating such conditions. However, due to its very short half-life in the bloodstream, frequent injections of this drug are required to maintain therapeutic drug concentration in the blood. This results in poor patient compliance and in oscillating systemic concentrations. This explains growing interest in drug administration via the skin for local as well as systemic therapy. The fact that Transfersomes®, can deliver small or large molecules through the skin barrier prompted us to design Transfersomes® loaded with SOD. These yielded encouraging first results. Our further investigations with acute and chronic inflammatory models have shown usefulness of transdermal delivery of Cu, Zn-SOD with Transfersomes® for inflammation therapy. We report here the results of enzyme biodistribution study done with epicutaneous application of SOD-loaded Transfersomes®. Subcutaneous administration was used for comparison to check and prove the efficacy of Transfersomes® mediated enzyme transfer through the skin. This work was partially financially supported by the research project POCTI/1999/FCB 35787. Prostaglandin E-1 is a potent vasodilator and platelet aggregation antagonist. In endothelial cells, PGE-1 has been shown to raise intracellular c-AMP levels, which results in the down-regulation of production of inflammatory cytokines (IFNγ, TNFαIL-2, etc.). The combined pharmacological effects of this drug make it an attractive candidate for the treatment of peripheral arterial disease and critical limb ischemia. However, the short half-life (approximately 90 s) and labile nature limit its efficacy. Here, we report the development of Liprostin™, a novel, multilamellar, freeze-dried liposome formulation containing PGE-1 that has been shown to increase the stability, and to prolong the half-life of PGE-1, when administered intravenously in both animals and healthy human subjects. To our knowledge, this is the first report of high i.v. boluses of PGE-1 (20 micrograms–120 micrograms) and well-tolerated infusion rates (one-hour at 6.0 micrograms/kg/hr which translated to 368 micrograms/h), and demonstrates the safety and potential efficacy afforded by the incorporation of this multipotent drug into a stable liposomal carrier. As a result of these findings and discussions with the U.S. Food and Drug Administration, Liprostin soon will be evaluated in Phase III clinical trials as an adjunct treatment along with angioplasty for critical limb ischemia. Liposomal formulations of some i.v. drugs or agents, including pegylated liposomal doxorubicin (Doxil, Caelyx), Ambisome and 99mTc-HYNIC PEG liposomes have been reported to cause hypersensitivity reactions (HSRs) in a considerable proportion (5–10%) of patients. The mechanism of these reactions have not been clarified to date. Our studies testing the hypothesis that complement (C) activation might play a causal role revealed the following facts: Doxil and PEG-PE containing placebo liposomes, phosphatidylglycerol (PG)-containing negatively charged vesicles and HYNIC PEG liposomes were potent C activators in human serum, whereas liposomes lacking PEG-PE or PG caused no C activation. The C activator liposomes also caused massive cardiopulmonary distress in pigs, whereas small neutral vesicles without PEG-PE, or PEG-PE micelles caused no hemodynamic changes. A clinical study in cancer patients showed increased plasma C terminal complex (SC5b-9) levels at 10 m postinfusion relative to baseline in 90% of patients displaying HSR and in 56% of non-reactor patients, with relative increases significantly greater in the reactor group. C activation and the presence of HSRs also showed positive correlation with Doxil dose. These data suggest that 1) C activation is due to the presence of PEG-PE and/or negative surface charge on liposomes; and 2) it might play a causal role in HSRs, although C activation does not necessarily cause clinical symptoms. Targeting the tumor vasculature in the treatment of cancer could be a very effective approach. Not only is the vascular bed the first target encountered by systemic injected drugs, it is also very important for the survival of the tumor. We demonstrated that destruction of the tumor associated vasculature (TAV), or manipulation of the TAV, to facilitate augmented extravazation of chemo-therapeutic agents, improved greatly the concomitant chemotherapy (). As the TAV is recognized as a major candidate in tumor therapy it is important to understand anti-vascular effects better. In our laboratory we examine the effect of immunotherapy (with for instance tumor necrosis factor alpha (TNF)) on the TAV, and the effect of anti-vascular therapy on tumors (). This is studied in animal models which exhibit similarities with the clinical setting. We observed that low-dose TNF improves tumor response dramatically when combined with Stealth liposomal doxorubicin (Doxil), which was accompanied by an augmented accumulation of doxorubicin in tumor tissue specifically. Secondly, the effect of TNF could be repeated using Stealth liposomal cisplatin (CIS-SL). In spite of the poor activity of the liposomal preparation, addition of TNF resulted in significant improved tumor response. We previously reported on the production of Stealth liposomal TNF (TNF-SL), which exhibits prolonged circulation time, reduced toxicity and augmented tumor localization compared to free TNF (). Recently we demonstrated that addition of TNF-SL to systemic treatment with DOXIL resulted in strong anti-tumor response (). Attempts are made to elucidate the mechanisms behind TNF-based therapies. Especially models which allow real-time monitoring of tumor vascular events in vivo are valuable in this type of research. One such model is the skin-fold window chamber. Using the window model we observed a heterogeneous distribution of systemically injected Doxil in tumors. As often total drug uptake is measured in tumors, this could result in misinterpretation of the efficacy of chemotherapy, as some tumor areas contain high amounts of drug whereas in other areas no accumulation of drug is observed. Ten Hagen TLM, van Der Veen AH, Nooijen PTGA, Van Tiel ST, Seynhaeve ALB, Eggermont AMM. Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats. Int J Cancer 2000; 87(6):829–837. de Wilt JHW, ten Hagen TLM, de Boeck G, van Tiel ST, de Bruijn EA, Eggermont AMM. Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion. Br J Cancer 2000; 82(5):1000–1003. van der Veen AH, de Wilt JHW, Eggermont AMM, van Tiel ST, Seynhaeve ALB, ten Hagen TLM. TNF-alpha augments intratumoral concentrations of doxorubicin in TNF-alpha-based isolated limb perfusion in rat sarcoma models and enhances anti-tumour effects. Br J Cancer 2000; 82(4):973–980. ten Hagen TLM, Eggermont AMM, Lejeune FJ. TNF is here to stay—revisited. Trends Immunol 2001; 22(3):127–129. van der Veen AH, Eggermont AMM, Seynhaeve ALB, van Tiel ST, ten Hagen TLM. Biodistribution and tumor localization of stealth liposomal tumor necrosis factor-alpha in soft tissue sarcoma bearing rats. Int J Cancer 1998; 77(6):901–906. ten Hagen TLM, Seynhaeve ALB, van Tiel ST, Ruiter DJ, Eggermont AMM. Pegylated liposomal tumor necrosis factor-alpha results in reduced toxicity and synergistic antitumor activity after systemic administration in combination with liposomal doxorubicin (Doxil) in soft tissue sarcoma-bearing rats. Int J Cancer 2002; 97(1):115–120. Photodynamic therapy (PDT) with photosensitizer and laser has been established as a potent and less invasive treatment for various tumors. Photofrin is a presentative photosensitizer used in PDT, but its distribution to skin causes photosensitive skin damage as side effects. In this study, we examined whether liposomalization of Photofrin increases accumulation of Photofrin in tumor and reduce side effects. Liposomal Photofrin (PF-Lip) induced accumulation of Photofrin in tumor and serum compared to Photofrin solution (PFsol). Namely, it was clear that liposomalization of Photofrin induced blood circulation and passive targeting of Photofrin. In skin, it was shown that Photofrin concentration in PF-Lip group decreased. However, after PF-Lip treatment, Photofrin concentration in liver increased to trap by reticuloendothelial system. While, when polyethyleneglycol modifid PF-Lip (PF-PEG-Lip) was administered, the Photofrin concentration in tumor was higher than that in PF-Lip group, and that in liver decreased to same level of PFsol. Furthermore, Irradiation with an excimer dye laser at 8 h after PF-Lip administration showed high efficacy of PDT, compared to that of PFsol. These results suggeted that liposomalization of Photofrin and PEG-modification of its liposome enhanced the therapeutic efficacy of PDT. [ABSTRACT FROM AUTHOR]

In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocytemacrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFVGM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFVGM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers. [ABSTRACT FROM AUTHOR]

We studied the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-[SUP3]H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000-54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. [ABSTRACT FROM AUTHOR]

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. 111In-labeled 34A-liposomes containing monosialoganglioside (GM1) were prepared that included [3H]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [3H]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of 111In-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen. Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors. [ABSTRACT FROM AUTHOR]

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.

We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In the last decades the mechanism of tumor cell killing by macrophages have been studied extensively. The tumor cytotoxic function of macrophages requires stimulation either with bacterial cell wall products such as lipopolysaccharide (LPS) or muramyldipeptide (MDP) or with cytokines such as interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated macrophages secrete several substances that are directly involved in tumor cell killing i.e. tumor necrosis factor (TNF) and nitric oxide (NO). On the other hand, substances are secreted that are able to stimulate tumor cell growth, depending on the stage and the nature of the tumor. Several clinical trials have been performed aiming at the activation of macrophages or dendritic cells, a subpopulation of the macrophages. In this review we will summarize and discuss experimental studies and clinical trials based on the activation of macrophages.

Purpose: Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases)., Methods: A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [3H]cholesteryloleylether or bilayer-anchored 5-fluoro[3H]deoxyuridine-dipalmitate ([3H]FUdR-dP) as a marker., Results: In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages., Conclusions: Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.

In this chapter we summarize literature and describe in more detail our own observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. Evidence is presented indicating that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in determining liposome accessibility of hepatocytes is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to postulate the existence of at least four different mechanisms of interaction of liposomes with hepatocytes, i.e. an endocytic and a non-endocytic one for both neutral and negatively charged vesicles

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) 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 (3)H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the (3)H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-(14)C-oleate as relative measure of degradation. Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3-4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes. In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface. These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.

Background: As a majority of ovarian cancer patients will ultimately develop recurrent disease, there is an urgent need for alternative or additional approaches in the treatment of this cancer., Materials and Methods: The antitumour effect of i.p. administered cisplatin, liposomal muramyltripeptide phosphatidylethanulamine (L-MTP-PE) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated using an i.p. growing murine ovarian tumour. Tumour growth was followed by measuring weight and survival of the mice., Results: An i.p. injection of L-MTP-PE in non-tumour bearing mice resulted in an approximately 10-fold increase in the number of peritoneal cells, which were highly cytotoxic. Nonetheless, treatment of mice inoculated with MOT cells with cisplatin, L-MTP-PE and GM-CSF using different treatment schedules did not result in inhibited tumour growth when compared to treatment with cisplatin alone., Conclusion: Although L-MTP-PE showed an enormous increase in peritoneal cells with high tumour cytotoxic capacity, the immunotherapeutic treatment with GM-CSF and L-MTP-PE, aimed at the recruitment and activation of the peritoneal cell population, failed to result in a significant prolongation of survival.

Sterically stabilized liposomes are considered promising carriers of therapeutic agents because they can facilitate controlled release of the drugs, thereby reducing drug-related toxicity and/or targeted delivery of drugs. Herein, we studied the pharmacokinetics and biodistribution of repeated injections of radiolabeled polyethyleneglycol (PEG) liposomes. Weekly injections of (99m)Tc-PEG liposomes dramatically influenced the circulatory half-life in rats. Biodistribution 4 h after the second dose showed a significantly reduced blood content (from 52.6 +/- 3.7 to 0.6 +/- 0.1% injected dose (ID), P <.01) accompanied by a highly increased uptake in the liver (from 8.1 +/- 0.8 to 46.2 +/- 9.8%ID, P <.01) and in the spleen (from 2.2 +/- 0.2 to 5.3 +/- 0.7%ID, P <.01). At subsequent injections the effect was less pronounced: after the fourth dose, the pharmacokinetics of the radiolabel had almost returned to normal. The same phenomenon was observed in a rhesus monkey, but not in mice. The enhanced blood clearance of the PEG liposomes also was observed in rats after transfusion of serum from rats that had received PEG liposomes 1 week earlier, indicating that the enhanced blood clearance was caused by a soluble serum factor. This serum factor was a heat-labile molecule that coeluted on a size exclusion column with a 150-kDa protein. In summary, i.v. administration of sterically stabilized PEG liposomes significantly altered the pharmacokinetic behavior of subsequently injected PEG liposomes in a time- and frequency-dependent manner. The observed phenomenon may have important implications for the repeated administration of sterically stabilized liposomes for targeted drug delivery.

Radiolabeled ([3H]cholesteryloleyl ether) immunoliposomes directed against rat colon adenocarcinoma CC531 cells were prepared by random coupling of a tumor cell-specific antibody, CC52, via a thio ether bond. In vitro binding experiments demonstrated a saturable and specific interaction of CC52-immunoliposomes, which could be inhibited by free non-coupled CC52 but not by irrelevant antibodies. The in vivo targeting potential of CC52-immunoliposomes, which were pegylated to achieve prolonged circulation times, was tested in an established rat liver CC531 metastasis model. Twenty-four hours after injection of the liposomes, 25% of the CC52-immunoliposomes were still present in the blood, which was comparable with the control liposomes (either with or without antibody). Liposomes were mainly taken up from the blood by the liver and the spleen, although hepatic uptake of the immunoliposomes was higher and splenic uptake was lower as compared to liposomes without antibody. Within the metastatic tumor nodules in the liver, uptake of both the CC52-immunoliposomes and non-specific immunoliposomes was significantly higher than that of control liposomes without antibody. Visualization of fluorescently or gold labeled CC52-immunoliposomes revealed that, although targeting to liver metastases was achieved, the immunoliposomes were mostly not associated with tumor cells but rather localized in tumor associated cells, probably macrophages.

Objective: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver., Study Design: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery., Results: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery., Conclusion: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

Liposomes of 400 nm in diameter can cross the 100-nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid-type but not solid-type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS-containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigated in vivo and in vitro. Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells, in vitro, do bind and internalize liposomes containing >/=30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells under in vivo conditions can be attributed to plasma effects since addition of various sera caused severe reduction of in vitro uptake of liposomes. In vivo the phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine., (Copyright 1999 Academic Press.)

Tumour-bearing rats were randomized to a 70% partial hepatectomy or a sham operation. At days 1, 3 or 14, portal and systemic serum was obtained and colon carcinoma cells were cultured in the presence of 5, 10, 20 or 50% serum. Proliferation and epidermal growth factor receptor (EGFr) expression was measured in tumour cells. Proliferation was 25-40% higher in tumour cells cultured with portal serum after hepatectomy than after sham operation when using serum obtained at day 3, but not days 1 and 14 after operation. In cultures with serum obtained at day 14 after operation CC 531 cells showed a 30% higher proliferation rate with systemic hepatectomy serum than CC 531 cells with sham systemic serum. These effects were not mediated by a change in EGFr mRNA and protein levels as the used colon carcinoma cells did not reveal EGFr activity by any of the three detection methods used.