4164 Nucleic Acids Research, 2008, Vol. 36, No. 12 demonstrated clear-cut enhanced pharmacological eﬀects of systemically administered siRNA as a result of use of delivery modalities. Some of these reports have been discussed above in terms of results at the cell culture level. Prior to initiating this discussion it is important to realize that there is a dichotomy in the behavior of oligonucleo- tides when comparing the in vivo situation to cell culture. Thus, almost without exception, eﬀective use of antisense or siRNA in cell culture requires a delivery agent such as a cationic lipid; in contrast, many of the successful in vivo studies with oligonucleotides have used ‘free’ compounds (7,60). There seem to be two possible interpretations of this dichotomy. One version suggests that cells undergo radical changes in organization and gene expression as they go from a 3D tissue environment to a 2D culture environment and that in this process key oligonucleotide transporters are lost. While it is clear that cells do undergo dramatic changes in the transition from 3D to 2D (141), it seems unlikely to us that the same oligonucleotide transport systems would be lost in every single type of cell. Another interpretation is a pharmacodynamic one. Because of experimental constraints, in culture cells are only brieﬂy exposed to the oligonucleotides; in contrast, most in vivo therapeutic experiments involve multiple doses and protracted exposures, thus perhaps allowing gradual intercellular accumulation of oligonucleotides. Surprisingly, this important dichotomy has not been carefully addressed via experimentation. Biological barriers to in vivo delivery of oligonucleotides In planning for the eﬀective delivery of oligonucleotides, it is essential to understand key aspects of endocytosis and intracellular traﬃcking at the cellular level. However, in vivo there are a number of other important parameters to consider as well (Figure 3). Essentially, a series of In Vivo Barriers to the Effective Delivery of Antisense and siRNA Oligonucleotides 1. Rapid excretion via the kidney 2. Degradation by serum and tissue nucleases 3. Uptake by the phagocytes of the reticuloendothelial system leading to sequestration in liver and spleen 4. Failure to cross the capillary endothelium 5. Slow diffusion/binding in extracellular matrix 6. Inefficient endocytosis by tissue cells 7. Inefficient release from endosomes Figure 3. In vivo barriers. The ﬁgure lists key barriers to eﬀective in vivo delivery of oligonucleotides. Rapid excretion is an issue for low molecular weight compounds. Clearance by phagocytes, capillary permeability and slow diﬀusion in the extracellular matrix apply to larger molecules and nanoparticles. Both small and large delivery agents can be aﬀected by poor cellular uptake and ineﬃcient release from endosomes. biological barriers stand between the newly administered oligonucleotide and its ultimate site of action in the cytosol or nucleus of tissue cells (142). For ‘free’ oligonucleotides or small conjugates, an important limita- tion is rapid excretion via the kidney. Molecules less than 5000 molecular weight are rapidly ultraﬁltered in the glomerulus and, in the absence of re-uptake, are accumu- lated in the urine (143). This picture is somewhat altered with oligonucleotides that bind strongly to plasma proteins thus retarding ultraﬁltration (144). The vascular endothelial wall comprises another major barrier, espe- cially for larger carriers. In general, molecules with a diameter of >45 A˚ (equivalent to about the size of an immunoglobulin) do not readily pass across the capillary endothelium and thus cannot eﬃciently enter the extra- cellular ﬂuid that bathes tissue cells (145). In addition, it is not only the size but also the shape of the macromolecule or nanocarrier that aﬀects its ability to traverse the endothelium (146,147). In a few tissues, including liver and spleen, the vascular endothelium is ‘fenestrated’ with gaps that allow the egress of macromolecules and nanoparticles up to about 200 nm diameter (148). In addition, work in xenograft tumors has given rise to the concept that the tumor vasculature is far ‘leakier’ than normal vasculature, thus also allowing egress of relatively large macromole- cules and nanoparticles, the so-called ‘EPR eﬀect’ (enhanced permeation and retention) (149). However, it is not clear that spontaneously occurring tumors in animals or humans uniformly display such increased leakiness. Further, tumors often exhibit other properties, such and increased interstitial pressure, that would tend to oppose delivery of nanocarriers to the tumor (150). Although one must be concerned about the issue of vascular permeability, it is also important to realize that the vascular endothelial cells themselves can be portals for therapy. This is especially true in sites of inﬂammation, where the endothelium upregulates key cell surface proteins including VCAM, ICAM and P-Selectin (151) or in angiogenic endothelium where the avb3 integrin is upregulated (152); thus, in both situations there is enhanced expression of receptors that can be addressed via CTLs linked directly to oligonucleotides or to nanocarriers. There are already many examples in the literature where endothelial receptors, especially avb3, have been targeted by various macromolecular or nanoparticle carriers bearing drugs or imaging agents (153–155). Even if a nanocarrier exits the vasculature, it still needs to diﬀuse through the extracellular matrix to reach tissue cells; the ECM is comprised of a dense meshwork of proteins and proteoglycans that can hinder nanocarrier diﬀusion and in some cases may even tightly bind the carrier (142). Another key barrier is presented by the phagocytes of the reticuloendothelial system (RES). These cells monitor the blood and remove foreign materials such bacteria and viruses; unfortunately, they also tend to treat administered macromolecules and nanoparticles as for- eign, thus accumulating these materials in hepatic Kupﬀer cells, splenic macrophages and other sites rich in phagocytes (156,157). This process can be attenuated to some degree by ‘passivating’ or ‘stabilizing’ the surfaces