Jan A. A. M. Kamps

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

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.

Molecular Pathways of Endothelial Cell Activation for (Targeted) Pharmacological Intervention of Chronic Inflammatory Diseases

In chronic inflammatory conditions, endothelial cells actively recruit immune cells from the circulation into the underlying tissue and participate in angiogenesis to support the continuous demand for oxygen and nutrients. They do so in response to activation by cytokines and growth factors such as tumour necrosis factor alpha (TNFalpha), interleukin-1 (IL-1), vascular endothelial growth factor (VEGF), and fibroblast growth factors (FGFs). Receptor triggering initiates intracellular signal transduction leading to activation of nuclear factor kappaB (NFkappaB), mitogen activated protein kinase (MAPK) activity, and nitric oxide and reactive oxygen species production, among others. As a result, adhesion molecules, cytokines and chemokines, and a variety of other genes are being expressed that mediate and control the inflammatory process. In recent years, different classes of drugs have been developed that interfere with selected enzymes involved in the intracellular signalling cascades. In endothelial cell cultures, they exert potent inhibitory effects on the expression of genes, while several studies also report on in vivo effectiveness to confine the inflammatory responses. To prevent undesired toxicity and to improve drug behaviour and efficacy, drug carrier systems have been developed that selectively deliver the therapeutics into the activated endothelial cells. The above subjects are recapitulated to give an overview on the status of development of endothelial cell directed therapeutic strategies to pharmacologically interfere with chronic inflammatory diseases.

BIODISTRIBUTION OF LIPOSOMES CONTAINING SYNTHETIC GALACTOSE-TERMINATED DIACYLGLYCERYL-POLY(ETHYLENEGLYCOL)S

We describe the synthesis of biodegradable poly(ethyleneglycol)-coupled galactolipids in which the galactose moiety is separated from a diacylglyceride lipid anchor by poly(ethylene glycol) chains of 10, 20 or 40 oxyethylene residues (PEG10/20/40). These Gal-PEG lipids (Gal-PEG-Lip) were incorporated in the bilayer of liposomes. The surface exposure of the galactose was investigated by aggregation experiments with ricinus communis agglutinin 120. Only the liposomes containing the PEG10 galactolipid aggregated with the lectin. Therefore liposomes were prepared containing Gal-PEG10-Lip and a trace amount of [3H]cholesteryl oleyl ether with an average diameter of approximately 100 nm and injected intravenously into rats. The Gal-PEG10-Lip liposomes were cleared from plasma with a T1/2 of 0.3 h. Identically sized and composed control liposomes without the Gal-PEG10-Lip had a T1/2 of approximately 12 h. The rapid plasma elimination of the Gal-PEG10-Lip liposomes could be attributed entirely to increased uptake by the liver amounting to more than 90% of injected dose. Uptake by the spleen was decreased to less than 1% of injected dose. A single injection of N-acetylgalactosamine 1 min prior to Gal-PEG-Lip liposome administration reduced the initial rate of plasma clearance to control levels. The increased liver uptake was almost entirely attributable to increased uptake by the Kupffer cells. Incorporation of PEG-DSPE in the Gal-PEG10-Lip liposomes only partially reversed the effect of the galactolipid with respect to liver and spleen uptake as well as intrahepatic distribution. These experiments demonstrate that liposome surface-exposed galactose residues, even if attached at the distal end of a poly(ethyleneglycol) chain anchored in the liposomal bilayer are effectively recognized by the galactose particle receptor on the Kupffer cells but fail to achieve significant targeting to the asialoglycoprotein receptor on the hepatocytes.

MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

The role of hepatocytes in the clearance of liposomes from the blood circulation

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

Uptake of long-circulating immunoliposomes, directed against colon adenocarcinoma cells, by liver metastases of colon cancer

Abstract 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.

Angiogenesis in Synchronous and Metachronous Colorectal Liver Metastases The Liver as a Permissive Soil

Objective:Resection of a primary colorectal carcinoma (CRC) can be accompanied by rapid outgrowth of liver metastases, suggesting a role for angiogenesis. The aim of this study is to investigate whether the presence of a primary CRC is associated with changes in angiogenic status and proliferation/apoptotic rate in synchronous liver metastases and/or adjacent liver parenchyma. Methods:Gene expression and localization of CD31, HIF-1&agr;, members of the vascular endothelial growth factor (VEGF) and Angiopoietin (Ang) system were studied using qRT-PCR and immunohistochemistry in colorectal liver metastases and nontumorous-adjacent liver parenchyma. Proliferation and apoptotic rate were quantified. Three groups of patients were included: (1) simultaneous resection of synchronous liver metastases and primary tumor (SS-group), (2) resection of synchronous liver metastases 3 to 12 months after resection of the primary tumor [late synchronous (LS-group)], and (3) resection of metachronous metastases >14 months after resection of the primary tumor (M-group). Results:In all 3 groups a higher expression of the angiogenic factors was encountered in adjacent liver parenchyma as compared to the metastases. VEGFR-2 gene expression was abundant in adjacent liver parenchyma in all 3 groups. VEGF-A and VEGFR-1 were prominent in adjacent parenchyma in the SS-group. The SS-group showed the highest Ang-2/Ang-1 ratio both in the metastases and the adjacent liver. This was accompanied by a high turnover of tumor cells. CONCLUSIONIn the presence of the primary tumor, the liver parenchyma adjacent to the synchronous liver metastases provides an angiogenic prosperous environment for metastatic tumor growth.

Interaction of differently designed immunoliposomes with colon cancer cells and Kupffer cells. An in vitro comparison.

AbstractPurpose. Evaluate the effectiveness of distal-end coupling of a tumor-specific antibody to liposomal polyethylene glycol (PEG) chains to improve target binding and reduce interference by macrophage uptake. Methods. Monoclonal antibody CC52, specific for CC531 rat colon carcinoma, was coupled to the bilayer of PEG-liposomes (type I) or to the distal end of bilayer-anchored PEG-chains (type II). Uptake of both (radiolabeled)liposome types by CC531 cells and rat liver macrophages was determined. Results. With increasing antibody density, both immunoliposome types showed increased binding to target cells, but type II liposomes displayed better target recognition than type I. Uptake by macrophages increased with antibody density for both liposome types. Lowest uptake by macrophages was found for type II liposomes at low antibody densities. Unexpectedly, not only for type I but also for type II liposomes, in which the antibody is coupled via its Fc moiety, uptake by macrophages was inhibited by aggregated IgG, indicating involvement of Fc receptors. Also polyinosinic acid, an inhibitor of scavenger receptors, reduced uptake of type II liposomes. Conclusion. Although distal end coupling of antibodies to bilayer-anchored PEG chains in liposomes through the Fc moiety enhances target cell binding, it does not prevent the recognition by Fc receptors on macrophages.

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 microg/micromol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.