Possibility of active targeting to tumor tissues with liposomes

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Abstract

In terms of active targeting by immunoliposomes, two anatomical compartments are considerable for targeting sites. One is located a readily accessible site in intravascular, and another is a much less accessible target site located in the extravascular. However, it was made clear that the active targeting with immunoliposomes is determined by two kinetically competing processes, such as binding to the target site and uptake by the RES. To overcome these contradictions, we have designed a new type of long-circulating immunoliposome, which was PEG-immunoliposome attached antibodies at the distal end of PEG chain, so called the pendant type immunoliposome. The pendant type immunoliposome showed much higher targetability than the ordinary immunoliposomes to both targeting sites of lung endothelial cells and solid tumor tissue. This is due to the free PEG chains (not linked to the antibody) effectively avoiding the RES uptake of liposomes, resulting in elevated the blood concentration and enhanced the target binding of immunoliposomes. The presence of free PEG does not interfere with the binding of the terminally linked antibody to the antigen. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to make the pendant type immunoliposomes (34A-PEG-ILP). 34A-PFG-ILP showed significantly higher targeting degree than the ordinary type of immunoliposomes. For targeting to the solid tumor tissue, Fab′ fragment of 21B2 antibody which is anti-human CFA and transferrin (TF) were used. Both pendant type immunoliposomes (Fab′-PFG-ILP and TF-PEG-ILP) showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. TF-PEG-ILP was internalized into tumor cells with receptor mediated endocytosis, after extravasation into tumor tissue. The pendant type immunoliposome can escape from the gaps between adjacent endothelial cells and openings at the vessel termini during tumor angiogenesis by passive convective transport much rather than ligand directed targeting. Active targeting to tumor tissue with the pendant type immunoliposome is particularly important for many highly toxic anticancer drugs for cancer chemotherapy. An ultimate goal of pendant type immunoliposome is the incorporation of a fusogenic molecule that would induce fusion of liposome following their binding to the target cells or their internalization by endocytosis. Such liposomal formulations should be useful for endocytotic internalization of plasmid DNA and other bioactive materials.

Introduction

It was German bacteriologist Paul Ehrlich who, in the late nineteenth century, coined the term “magic bullet”, meaning a chemical that travels through the body and selectively kills diseased cells without harming neighboring healthy ones [1]. Drug delivery with “magic bullet” to cells, tissues or organs is defined as “active targeting”. Many different approaches using various physical and biochemical principles have been proposed and examined to develop a system to achieve a therapeutically acceptable degree of target specificity [2], [3], [4], [5]. With the availability of a suitable ligands, this approach takes advantage of a relatively abundant expression of a particular receptor on the target cell.

Among the different approaches of active targeting, immunoliposomes using an antibody as a targeting ligand and a lipid vesicle as a carrier for both hydrophobic and hydrophilic drugs, have attracted much attention [6], [7]. The rationale of this approach has been well established in various in vitro systems. The use of an antibody molecule as a homing device has been especially facilitated by the development of the hybridoma technology, which makes it possible to produce a large quantity of a monoclonal antibody to a wide variety of cell determinants [8]. However, only a limited number of preclinical studies report successful targeting of immunoliposomes in vivo [9]. Studies in vivo have revealed that coating liposomes with antibody leads to enhanced uptake of the immunoliposomes by the reticuloendothelial system [10], [11] and the immunotargeting efficiency depends on the antibody density on the surface [12]. Thus, highly efficient targeting and a relatively low level of RES uptake of immunoliposomes are apparently mutually exclusive.

Given a suitable antibody with high specificity and affinity for the target antigen, the critical factor is the accessibility of target cells to immunoliposomes. Efficient target binding of the injected immunoliposomes occurs only when the target cell is in the intravascular compartment or can be accessible through leaky vascular structures. Thus, in terms of targeting drug delivery by immunoliposomes, two anatomical compartments are considerable for targeting sites. One is located at a readily accessible site in intravascular, such as the vascular endothelial surface, T cells, B cells or thrombus. Another is a much less accessible target site located in the extravascular. This site involves a solid tumor, an infection site, or an inflammation site, which vascular structure is leaky.

In this review, we describe the current status of the active targeting by immunoliposomes. To characterize the parameters for such immunoliposome targeting in vivo, two different targeting models in mice were employed; the mouse lung targeting model for a readily accessible site and the implanted mouse solid tumor model for a less accessible site located in extravascular. We discuss its associated problems and future perspectives, with emphasis being placed on applications of immunoliposomes in vivo.

Section snippets

Anatomical barriers of tumor tissue

In general, tumor vessels are inherently leaky, due to the wide inter-endothelial junctions, large number of fenestrae and transendothelial channels formed by vesicles, and discontinuous or absent basement membrane [13], [14]. In contrast, the blood vessels in most normal tissues are nonfenestrated capillaries. These blood vessels are composed of a single layer of endothelial cell with tight junctions. The endothelial barrier may prevent liposomes to traverse intact vessels. The cartoon of

Rapid uptake of immunoliposomes by reticuloendothelial system (RES)

Liposomes have been proposed to be useful drug carriers for targeted drug delivery systems, and are under investigation in several therapeutic fields [18], [19]. In order to achieve maximum targeting, liposomes should remain in the systemic circulation for a relatively long period of time. However, formulations of liposomes used in the past were rapidly removed from the circulation by the reticuloendothelial system (RES) [20], [21]. Furthermore, it is known that coating liposomes with

Long-circulating (RES-avoiding) liposome and passive targeting to tumor tissue

It has been demonstrated recently that newly developed liposomes, containing either monosialoganglioside GM1 (GM1) [22] or amphipathic polyethylene glycol (PEG) derivatives [23], [24], [25], are not readily taken up by the macrophages in the RES and hence stay in the circulation for a relatively long period of time (Fig. 2). Particularly, PEG is useful because of its ease of preparation, relatively low cost, controllability of molecular weight and linkability to lipids or protein including the

Pendant type immunoliposome design

Immunoliposomes for the treatment of tumor should satisfy a number of requirements aimed at maximum targeting effect of immunoliposome administered systemically in the bloodstream. Antigen binding site of the liposome-conjugated antibody must be accessible for unperturbed interaction with antigen on the surface of target cells. The blood clearance of immunoliposomes must be minimized in comparison with rate of extravasation in the tumor. Immunoliposome must allow efficient loading and retention

Anatomical barriers

The blood vessels in most normal tissue are nonfenestrated capillaries. Given a suitable antibody with high specificity and affinity for the target antigen, the critical factor is the accessibility of immunoliposomes to the target cells localized on the other side of the endothelium. Another potential approach from described here is utilization of the transcytosis function of endothelial cells [36]. In transcytosis, certain ligand–receptor complexes are, upon endocytosis, transported across the

Conclusion

We have designed a new type of long-circulating immunoliposome, which were PEG-immunoliposomes attached antibodies at the distal end of PEG chain, so called the pendant type immunoliposome, and discussed the active targeting to the two different anatomical compartments by the pendant type immunoliposomes. One is located a readily accessible site in intravascular, such as pulmonary endothelial cells, and another is a much less accessible target site located in the extravascular, such as solid

Abbreviations

    21B2

    monoclonal antibody IgG1 antibody, specific for the human carcinoembryonic antigen

    CEA

    human carcinoembryonic antigen

    Chol

    cholesterol

    DiI

    1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

    DPPC

    dipalmitoyl phosphatidylcholine

    DPPE-PEG-Mal

    dipalmitoyl phosphatidylethanolamine derivative of PEG with a terminal maleimidyl group

    DSPC

    distearoyl phosphatidylethanolamine

    DSPE

    distearoyl phosphatidylcholine

    DSPE-PEG

    distearoyl-N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine

Acknowledgements

Work done in this laboratory is supported by the Grant-Aid for Scientific Research (No. 04671332, 08672568 and 10470254) from the Ministry of Education, Science and Culture, and the Grant-Aid for Cancer Research (No. 9-Specified) from the Ministry of Health and Welfare.

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