4166 Nucleic Acids Research, 2008, Vol. 36, No. 12 complexation with siRNA (109). This ‘nanocomplex’ approach was used to deliver growth inhibitory siRNAs in vivo to B16 melanoma cells engineered to express the HIV envelop glycoprotein, or to breast tumor cells over- expressing ErbB2, using the appropriate antibody com- ponent in each case. A modiﬁcation of this approach (110) used a protamine chimera with a conformation-sensitive single chain antibody directed against the LFA-1 integrin to speciﬁcally target siRNA in vivo to lymphoid tumor cells that express the activated form of LFA-1 (110). Polymer systems have also been used for targeted in vivo delivery. Thus, as mentioned earlier, a polymer nanocar- rier linked to N-acetylgalactosamine was used to promote selective uptake by hepatocytes (113). In vivo, this system was able to eﬀectively deliver apoB siRNA to mouse liver, resulting in gene silencing and reduced systemic choles- terol levels. Somewhat similarly, a system involving an RGD-conjugate of PEI was used to deliver siRNA targeting VEGF-receptor to tumor vasculature (125). Finally, as mentioned earlier, delivery of siRNA to the brain was attained by formation of a nanocomplex with a peptide comprising positively charged sequence to bind the oligonucleotide and a sequence that binds with high aﬃnity to the nicotinic acetycholine receptor in neurons (114). Use of an antiviral siRNA provided protection against a potentially fatal viral encephalitis in mice. It is very surprising that the siRNA nanocomplex was able to cross the blood–brain barrier since even most small molecule drugs fail to do so. However, in this case it is possible that the presence of an active viral infection altered the permeability of the barrier. In summary, several types of nanoparticle technologies have eﬀectively delivered siRNAs to the liver, to tumors, or to inﬂammed tissues. It should be noted, however, that the vasculature in these tissues diﬀers substantially from that more generally present in the body, as has been discussed earlier. Thus, it is unclear whether these approaches can be generalized to other tissues. Several of the studies mentioned above have also been reviewed recently in another venue (171). CONCLUSIONS As reﬂected in this article, a great deal of eﬀort is currently focused on the delivery of therapeutic oligonucleotides. Signiﬁcant strides have been made, but the issue has not been fully resolved. Thus, it seems valuable to compare and contrast in broad terms the various strategies that are being pursued. Perhaps the most signiﬁcant parameter to consider is the size of the moiety being delivered, contrasting nanoparticles that have diameters in the 50–200 nm range and molecular weights in the millions to monomolecular oligonucleotide conjugates with mole- cular weights in the thousands. Another key issue is charge since both polyanionic and polycationic molecules or nanocarriers can interact with blood proteins to incite toxicity or aﬀect biodistribution (172,173). Nanoparticles of various types oﬀer many advantages as delivery agents. They can carry a large ‘payload’ comprising hundred or thousands of copies of the siRNA or antisense oligonucleotide. They can be decorated with multiple copies of targeting ligands, thus providing high- avidity interaction with the target cells. Nanoparticles can be designed to release their contents at prescribed rates and can also be engineered to assist in the release of their contents from endosomes. Novel approaches for produ- cing extremely uniform nanoparticles with controllable drug release characteristics are becoming available (174). Technologies for producing nanoparticles are reasonably mature thus permitting relatively facile scale-up for clinical studies (although reliable large-scale formulation of nanoparticles under GMP conditions is not without problems). Additionally, regulatory agencies are familiar with nanoparticles as delivery agents, based in part on the several liposomal drugs now on the market (175). Many of these useful aspects of nanoparticle-mediated oligonucleo- tide delivery are implicit in the studies reviewed earlier. However, the many positive features of nanoparticles are counterbalanced by some important negative ones. First, despite advances in using PEG or other hydrophilic polymers for stabilization, a large fraction of the injected dose of nanoparticles will accumulate in the liver, and much of that will be taken up by hepatic phagocytes. Thus, a signiﬁcant portion of the dose of oligonucleotide will wind up at the wrong site, where toxic eﬀects could potentially occur. Second, because of the vascular endothelial barrier, nanoparticles can only reach certain tissues, such as liver and spleen, where gaps in endothe- lium occur. This is one of the reasons that the various siRNA and antisense companies have focused so strongly on diseases that involve the liver. Nanoparticles that have long circulation times can also accumulate in some types of tumors due to the EPR eﬀect; inclusion of a ligand that binds a receptor on the tumor cells can then promote uptake and intracellular delivery (176). However, what nanoparticles cannot do is to access parenchymal cells in most normal tissues; they are simply excluded by the endothelial barrier. Thus, many potential disease targets will not be addressable by oligonucleotide-based thera- peutic platforms that involve nanoparticles. In contrast, oligonucleotide conjugates are usually far smaller than the pores in normal vascular endothelium; thus, in principle, they should be able to access virtually all tissues, just as conventional drugs do (with the exception of the central nervous system). It should be noted that in some cases the oligonucleotide conjugate may be rapidly excreted via the kidney. Clearly, the detailed physical and chemical properties of individual oligonucleotide conju- gates will inﬂuence their interactions with plasma proteins and their overall biodistribution; however, to a ﬁrst approximation, the relatively small size of oligonucleotide conjugates implies a fundamental diﬀerence in their in vivo behavior as compared to nanoparticles. Nonetheless, there are some liabilities associated with this approach. First, each conjugate requires a separate synthesis, whereas a particular type of nanoparticle can potentially accomodate a variety of diﬀerent oligonucleotides. Second, since only a single ligand is conjugated to the oligonucleotide this implies a lower-aﬃnity interaction with target receptors than is the case for multivalent nanoparticles. Another issue concerns release from endosomes subsequent to