Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid–PEG conjugates

Abstract

Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.

Introduction

The rationale for cancer targeting with folate ligands attached to the liposome surface is based on two layers. First, there is a common layer to all folate-targeted systems which relates to the choice of the tumor cell folate receptor (FR) as the target. FR upregulation or over-expression is commonly associated with a broad variety of tumor types including solid and hematological malignancies, and it appears to be more frequently observed in advanced stages of cancer [1]. How specific and frequent is FR overexpression in cancer cells to justify its choice as target is discussed in other articles of this issue of Adv. Drug Deliv. Rev. and will not be addressed here. The second layer contains elements unique to liposomal and perhaps other nanoparticulate drug carrier systems and will be addressed here. The strength of the folate-targeted liposome approach stems from conceptual advantages over two alternative approaches: nontargeted liposomal systems and nonliposome-based folate-targeted systems.

A schematic illustration of the folate liposome targeting concept is presented in Fig. 1. Long-circulating liposomes, such as polyethylene glycol (PEG) coated liposomes (also known as STEALTH® liposomes) [2], tend to accumulate in tumors as a result of increased microvascular permeability and defective lymphatic drainage, a process also referred to as the enhanced permeability and retention (EPR) effect [3]. This is a passive and nonspecific process of liposome extravasation that is statistically improved by the prolonged residence time of liposomes in circulation and repeated passages through the tumor microvascular bed [4]. However, except for rare instances, tumor cells are not directly exposed to the blood stream. Therefore, for an intra-vascular targeting device to access the tumor cell FR, it must first cross the vascular endothelium and diffuse into the interstitial fluid. Experimental data with antibody-targeted liposomes and FTL indicate that liposome deposition in tumors is similar for both targeted and nontargeted systems [5], [6], [7], supporting the hypothesis that extravasation is indeed the rate-limiting step of liposome accumulation in tumors. However, once liposomes have penetrated the tumor interstitial fluid, binding of targeted liposomes to FR may occur thus shifting the intra-tumor distribution from the extracellular compartment to the tumor cell compartment, as shown recently for a mouse ascitic tumor [7]. Binding to tumor cells may be followed by internalization of liposome contents via folate-receptor mediated endocytosis (Fig. 1). Retrograde movement of liposomes into the blood stream, if any, will be reduced for liposomes with binding affinity to a tumor cell receptor. Obviously, when the parameter of drug delivery is considered, there will always be a combination of in situ release from an extracellular liposome depot and intra-cellular release from internalized liposomes. Therefore, the theoretical advantages of FTL over NTL are related to a shift of liposome distribution to the tumor cell compartment, delivery of liposomal contents to an intra-cellular compartment in liposome-associated form, and, possibly, prolonged liposome retention in the tumor. On the negative side, the main disadvantage of a targeted system to a cancer cell receptor such as FR is the difficulty of a large nanosize assembly, such as FTL, to penetrate a solid tumor mass, specially considering the high interstitial fluid pressure that is often present in tumor masses of clinically detectable size [8].

Liposomal systems offer an elegant drug delivery amplification system. Each liposome vesicle carries a drug cargo usually in the order of 10³–10⁴ molecules. For instance, in the case of a STEALTH® liposome formulation known as Doxil, there are between 10,000 and 15,000 doxorubicin molecules per vesicle [9], and these may be targeted with the help of as few ligands as 10 per vesicle, i.e. a 100–1000-fold delivery amplification factor when the drug:ligand ratio is considered. Another theoretical advantage of liposomal systems is that their size far exceeds the critical glomerular filtration threshold. Therefore, unlike low molecular weight folate-targeted complexes, FTL do not have access to the luminal side of kidney tubular cells where FR is expressed, thereby sparing kidneys of massive FR-mediated liposomal drug delivery and subsequent toxicity [10]. One of the disadvantages of FTL vis-à-vis small drug–folate conjugates is that liposomes are bulky structures that are difficult to internalize by nonphagocytic cells. The best characterized pathway of liposome internalization, mediated by clathrin-coated pits, often leads to sequestration of liposome contents within the lysosome compartment. An alternative pathway of endocytosis, known as potocytosis, may operate for receptors associated with cell caveolae or lipid rafts, such as FR [10], and facilitate access to the cytosol via acidic endosomes bypassing lysosomes. It is well established that FTL enter cells by FR-mediated endocytosis (FRME) [11]. In addition, experimental data with folate-targeted, pH-sensitive liposomes are consistent with liposome transit through an acidic vesicle compartment [12]. A connection between post-caveolar or post-raft endosomes and lysosomes is possible, since markers of the clathrin-coated pit pathway and folate conjugates have been shown to co-localize in the same cell organelles [13]. Thus, an important fraction of internalized liposomes may end up in lysosomes. The cell trafficking of liposomes following FRME needs to be better understood, specially since intra-cellular trafficking of small molecular weight folate conjugates may be different from that of nanoparticles with multimeric binding such as FTL.