Cellular Delivery Applications

Cellular Delivery Applications

For in vivo studies, it is important to keep in mind that oligonucleotides, and their delivery agents, almost always are taken up by the cell via endocytosis, and need to exit from the endosome to reach their designated cellular target. Thus, in order to obtain both good uptake and effective delivery of an oligo to its target in vivo, the multiple routes of endocytosis and the molecular trafficking pathways of cells need to be considered (1), as well as clearance issues at the whole organism level (for example, excretion by the kidney or uptake by phagocytes that may sequester the oligo in the spleen or liver) (2). Conjugation of cholesterol to siRNA or anti-sense oligos increases the resulting conjugate's association with serum proteins, which often has a positive effect on their pharmacokinetics (for example, elimination half-life and tissue accumulation), biodistribution and effects on gene expression (3). For cholesterol-linked siRNAs, both high density lipoprotein (HDL) and low density lipoprotein (LDL) were their primary carriers in serum. Uptake of the oligos occurs via both LDL- and HDL-receptors, with LDL-receptors being the predominant receptor utilized in liver (4). Cell entry appears to occur by a complex receptor-mediated endocytosis mechanism that includes transfer of the cholesterol-oligo conjugate from the receptor to a plasma membrane protein Sid1 along the way (4). Oligos can also be conjugated with PEG (PEGylated) to improve in vivo uptake. PEGylated oligo conjugates are less susceptible to phagocytes, since they poorly absorb to plasma proteins (opsonins) that enhance phagocytosis. This significantly increases the elimination half-life of the oligos in circulation, thereby facilitating cellular uptake (5).

References

(1) Juliano, R., Alam, Md.R., Dixit, V., Kang, H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. (2008), 36: 4158-4171.
(2) Juliano, R.L. Biological Barriers to Nanocarrier-Mediated Delivery of Therapeutic and Imaging Agents. In: Niemeyer, C.M., Mirkin, C.A., editors. Nanobiotechnology II. Weinheim, Germany: Wiley-VCH; 2007. pp. 263-278.
(3) Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature (2004), 432: 173-178.
(4) Wolfrum, C., Shi, S., Jayaprakash, K.N., Jayaraman, M., Wang, G., Pandey, R.K., Rajeev, K.G., Nakayama, T., Charrise, K., Ndungo, E.M., et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. (2007), 25: 1149-1157.
(5) van Vlerken, L.E., Vyas, T.K., Amiji, M.M. Poly(ethylene-glycol)-modified nanocarriers for tumor-targeted and intracellular delivery. Pharm. Res. (2007), 24: 1405-1414.
(6) Debart, F., Abes, S., Deglane, G., Moulton, H.M., Clair, P., Gait, M.J., Vasseur, J., Lebleu, B. Chemical Modifications to Improve the Cellular Uptake of Oligonucleotides. Curr. Top. Med. Chem. (2007), 7: 727-737.
(7) Manoharan, M. Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug Dev. (2002), 12: 103-128.
(8) Chaltin, P., Margineanu, A., Marchand, D., Van Aerschot, A., et al. Delivery of antisense oligonucleotides using cholesterol-modified sense dendrimers and cationic lipids. Bioconj. Chem. (2005), 16: 827-836.
(9) Lorenz, C., Hadwiger, P., John, M., Vornlocher, H.P., Unverzagt, C. Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. Bioorg. Med. Chem. Lett. (2004), 14: 4975-4977.