Cellular Delivery Design and Protocols

Cellular Delivery Design / Protocol

Unmodified oligonucleotides generally show poor bioavailability in vivo, due to their inability to effectively penetrate cell membranes. The primary reason for this is that the polyanionic character of unmodified oligonucleotides makes them highly hydrophilic, but since the surfaces of cell membranes are hydrophobic, the former cannot effectively enter into the cell.

To reduce this polyanionic effect and improve delivery efficacy, one approach is to use an oligonucleotide complexing agent as a delivery vehicle, for example, cationic lipid formulations, cyclodextrins, etc. Such complexing agents have been shown to increase the efficiency of oligo cellular uptake relative to that observed with naked oligonucleotides, but because the agent is not covalently bound to the oligo and an excess of the agent must be used, the actual amount of oligo that is actually available for delivery into the cell typically is low (6).

Alternatively, oligonucleotides can be directly conjugated to a lipophilic moiety that will make it easier for the oligo to penetrate cell membranes. Cholesterol and polyethylene glycol (PEG) are two such moieties that are commonly used as modifications for this purpose (7). An important advantage of this approach is that the amounts of lipophile-oligo conjugates actually available for delivery into the cell tend to be significantly higher than those observed with oligo-complexing delivery vehicles. At the same time, it is important to be aware that, in some cases, covalent attachment of the lipophilic moiety to the oligo negatively impacts the latter's intra-cellular distribution or ability to hybridize to its target. In such cases, a combination of the two approaches (complexation and direct conjugation) may be necessary to resolve the problem (6).

A related approach is to conjugate the lipophilic moiety to a "delivery" oligo that is complementary to the oligo slated for delivery into the cell. In duplex form, the modified "delivery" oligo thus serves to assist passage of the other oligo through the cell membrane. The effect is often enhanced when the duplex is mixed with a cationic lipid formulation or other oligo complexing agent before either being applied to cell culture or injected into the organism. An attractive feature of this "oligo-assisted oligo delivery" method is that because the lipophilic moiety is not conjugated to the oligo slated for delivery, it does not affect either that oligo's intra-cellular distribution or its ability to hybridize to its target. This strategy has been employed, using cholesterol as the lipophilic moiety, in both anti-sense and siRNA applications (8, 9).

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.