4160 Nucleic Acids Research, 2008, Vol. 36, No. 12 cholesterol (46,47). Initial uptake of receptor and ligand is followed by sequential intracellular traﬃcking into a variety of low pH endomembrane compartments, includ- ing early/sorting endosomes, late endosomes/multi- vesicular bodies, and lysosomes; in some cases, recep- tors/ligands can traﬃc to the Golgi complex. In many instances, receptor and ligand are dissociated in the low pH endosome environment, and in some cases the receptor can recycle back to the cell surface via the sorting endosomes (Figure 1). The complex ﬂow of endomembrane traﬃc (48) is regulated by the Rab family of small GTPases and by tethering complexes, while vesicular fusion events are controlled by SNARE proteins (49–51). The SNX (sorting nexin) proteins also are important in sorting and cargo retrieval from endosomes (52). The traﬃcking of inter- nalized receptor and ligand can often be very complex and dependent on the nature of the receptor, the physiological status of the cell and the cell type. Some receptors can enter cells via multiple pathways, for example, via both clathrin-coated vesicles and caveolae (53). The route of entry can aﬀect the ultimate fate and function of the ligand–receptor complex. For example, at low ligand levels the EGF-receptor is internalized via coated pits and can continue to signal, while at high ligand concentrations the receptor is ubiquitinated, internalized via a CLIC or caveolar pathway, and degraded (54). There are a number of tools commonly used to probe pathways of internalization. For example, many studies of oligonucleotide uptake have used pharmacological inhib- itors that putatively block speciﬁc endocytotic pathways. In reality, however, such inhibitors often aﬀect multiple uptake pathways, as well as having many other eﬀects on the cell. For example, although b-cyclodextrin is often used to block uptake involving lipid rafts, it also aﬀects clathrin-mediated endocytosis (55). Likewise, agents that block macropinocytosis by aﬀecting actinomyosin func- tion will also block other endocytotic pathways, as well as having generalized eﬀects on the cytoskeleton (56,57). Thus, results obtained with pharmacological inhibitors need to be interpreted conservatively. Potentially, more powerful approaches include using antibodies to key marker proteins to co-visualize ﬂuorophore-tagged oligo- nucleotides in speciﬁc endomembrane compartments (58), and use of dominant-negative Rab proteins to interfere selectively with traﬃcking patterns (59). Although general aspects of the cellular uptake of oligo- nucleotides have been studied for a long time and extensively reviewed (7,60,61), it is only recently that investigators have probed oligonucleotide internalization in light of current understanding of endosomal traﬃcking pathways. While not enough work has been done to achieve a broad consensus, some interesting examples of more detailed traﬃcking studies have recently emerged (62–64). Ligands for enhancing and targeting delivery In this section, we will examine ligands that have been used to attain cell-type selective targeting or to enhance the uptake of oligonucleotides. For the sake of simplicity, we have divided the discussion into agents that target speciﬁc receptors (CTLs, cell targeting ligands) and agents that enhance transmembrane permeation (primarily cell penetrating peptides, CPPs). actin Macropinosome Golgi apparatus Pinosome TGN230 Nucleus Endoplasmic reticulum LAMP-1 Lysosome MULTIPLE ENDOCYTOTIC UPTAKE PATHWAYS Macropinocytosis Smooth vesicle pinocytosis Clathrin mediated endocytosis Caveolar mediated uptake Clathrin coated pit CAVEOLIN-1 X Clathrin coated X X X X X XX Caveosomes vesicle XX X X CLAXTHRIN X XX EEA-1 eEnadrolys/osmoretsing Late endosome Figure 1. Endocytotic pathways. The ﬁgure depicts the multiple endocytotic pathways that may be involved in uptake of oligonucleotides. The black arrows represent well-documented traﬃcking routes within the cell. The names in red indicate well-known protein markers for various endomembrane compartments; in most cases, antibodies to these marker proteins are commercially available.