Cell mechanisms of liposome uptake

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. As previously described, the

rate limiting steps in this process are the efficiency of cell surface association, internalization, release of loaded drug/genes from intracellular compartments such as endosomes, transfer to the cytosol and eventually, translocation into the nucleus.

Liposomal carriers can adsorb specifically and non-specifically to the cell surface (Figure 1.4 a, b), in some cases promoting a direct delivery of the therapeutic agent into the cytoplasm (Figure 1.4 c, d). Additionally, in order to increase the initial cell binding, delivery carriers have been modified with cationic moieties to increase the electrostatic interaction of the vehicle with the negatively charged cell membrane (Figure 1.4 e, c). Constructs that are more sophisticated involve the covalent binding of a ligand or antibody to the liposome carrier to promote the specific receptor-mediated or antigen-mediated endocytosis, respectively (Figure 1.4 a). If uptake occurs via endocytosis (Figure 1.4 f), receptor or non-receptor mediated, the liposomes end up in endosomal compartments.

In the case of receptor-mediated endocytosis, internalized vesicles and their content are sorted mainly to the lysosomal compartment for degradation of the ligands and the loaded cargo (Figure 1.4 f, g). This also accomplishes the downregulation of activated receptors (Shepherd, 1989). However, the majority of targeted deliveries aim to avoid lysosomal trafficking in an effort to protect the drug molecule or biomolecules from enzymatic degradation.

Lysosomes, as well as the endoplasmatic reticulum (ER), Golgi apparatus, and endosomes are, to a variable degree topologically continuous with the exterior of the cell, i.e. the exit of macromolecules from any of these compartments requires passage through a lipid bilayer.

In general, the endocytosis mechanisms can be divided into clathrin-dependent and clathrin-independent. There is a close relation between particle size and the preferred internalization pathway, although the correlation is not absolute. Rejman et al. proposed that particles <200 nm are taken up via the clathrin-dependent mechanism whereas larger particles (250-500 nm) enter the cell through caveolin 1-rich vesicles (Rejman et al., 2004). This correlation, between particle size and uptake pathway, is indeed plausible since some of these pathways are constrained to the size of their vesicles – e. g. clathrin-coated and caveolae-derived vesicles (Pelkmans et al., 2004).

Drug or DNA

Figure 1.4: Liposome uptake and intracellular fate: DNA or drug loaded liposomes can specifically (a) or nonspecifically (b) adsorb onto the cell surface. Liposomes can also fuse with the cell membrane (c), and release their content into the cytoplasm, or can be destabilized by certain cell membrane components when adsorbed on the surface (d) so that the released drug can enter the cell via micropinocytosis. Liposomes can undergo the direct transfer or protein-mediated exchange of lipid components with the cell membrane (e) or be subjected to a specific or nonspecific endocytosis (f). In the case of endocytosis, liposomes can be directed into lysosomes (g) or, in route to the lysosome, they can provoke endosome destabilization (h), which results in the liberation of the therapeutic agent into the cell cytoplasm (Adapted from (Torchilin, 2005).

Clathrin-mediated endocytosis serves as the main mechanism of internalization for macromolecules and plasma membrane constituents for most cell types and it is the most investigated vesicular pathway for targeted drug delivery. This is initiated by the formation of clathrin-coated pits, which are subsequently pinched off and internalized. The clathrin coat is removed and multiple vesicles fuse originating the early and late endosomes that ultimately fuse with lysosomes (Mousavi et al., 2004).

1.4.1. Receptor-mediated endocytosis as a route for targeted liposomal delivery

Targeting is usually achieved by conjugating a high affinity ligand to the carrier that provides preferential accumulation of the latter for instance, in a tumor-bearing organ, in the tumor itself, in individual cancer cells or intracellular organelles. In most cases the targeting moieties (ligands or antibodies) are directed toward specific receptors or antigens exposed

on the plasma membrane (Kato and Sugiyama, 1997). The overexpression of receptors or antigens in many human cancers lends itself to efficient drug uptake via receptor-mediated endocytosis. Folate and Transferrin are widely applied ligands for liposome targeting because their cognate receptors are frequently overexpressed in a range of tumour cells (Kakudo et al., 2004; Hilgenbrink and Low, 2005). Liposomes tagged with various monoclonal antibodies have been delivered to many targets (Park et al., 2001).

The performance of non-viral vector could be certainly optimized by targeting them into distinct cellular internalization pathways, considering that not every pathway may be equally effective in releasing a therapeutic biomolecule in the cytosol. This step is critical for nucleic acid delivery to increase the possibility of nuclear transport and the ultimate expression of the delivered genes (Bareford and Swaan, 2007).

1.4.2. EGF receptor targeted delivery systems

The epidermal growth factor receptor (EGFR, ErbB) is a 170 kDa protein that is distributed randomly on the surface of cells, excluding hematopoietic cells. Its ligand, EGF, is a 53 aminoacid-peptide that mediates cellular signal events regulating cell proliferation, differentiation, cell cycle progression, adhesion, invasion, angiogenesis and inhibition of apoptosis. Following binding of the EGF, the EGFR functions either as a homodimer through the complexation of two identical EGFR molecules or as an heterodimer by associating with one of the three other ErbB family members; in either case the resulting dimerization then initiates the cellular internalization (Bublil and Yarden, 2007).

The EGFR is a tempting target for gene delivery since it is overexpressed in a wide variety of human tumors found in cancers of head and neck, breast, colon, ovary, lung, prostate and liver (Johnston et al., 2006). Enhanced EGFR expression is associated with tumor invasiveness, resistance to chemotherapy and radiation therapy and clinically correlates with poor prognosis and lower patient survival (Rubin Grandis and V. A.; Wagener, 1998).

Squamous cell carcinoma of the head and neck is a cancer commonly associated with EGFR overexpression (>90%), which appears to play a role in the unregulated growth of these cells (Cohen, 2006). EGFR has been used to target drugs and toxins loaded in therapeutic liposomes (Kullberg et al., 2003; Mamot et al., 2005) and lipoplexes (Shir et al., 2006).