**6. Cleavable peptide-phospholipid conjugates under physiological conditions**

Detachable conjugates between cationic and hydrophobic lipid portions may facilitate the DNA release from complexes in intracellular reductive environment. This is a promising strategy for improvement of efficacy of non-viral gene delivery. Such approaches have been reported using disulfide-linked polymer (Oba et al., 2008) and gemini lipids (Behr). Recently, we have successfully synthesized oligo-arginine bearing phospholipids via disulfide linkage, DPPE-SS-R*n* (Scheme 2). Oligo-arginines, e.g., TAT peptide, are well known for effective carriers across cell membranes (Futaki et al., 2001). The reaction scheme is shown in Scheme 2. In brief, 1,2-dipalmitoyl-*sn*-glycero-3-phospho-ethanolamine (DPPE) was reacted with a heterobifunctional coupling agent, *N*-succinimidyl-3-(2-pyridyldithio)-propionate

Polyamine – Lipid Conjugates as Effective Gene Carriers:

are effective for systemic siRNA delivery (Asai et al., 2001).

**8. Acknowledgments**

of the Japanese Government.

*Ed.* 42, 3153–3158.

*Bioconjugate Chem.* 22, 429–435.

*Acad. Sci. U.S.A.* 86, 6982–6986.

**9. References** 

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Chemical Structure, Morphology, and Gene Transfer Activity 261

pH-dependent transformation is crucial in gene transfer; and the chemical structure of the polyamine portion may therefore play an important role in the acidification-induced transformation. We have also described the relation between the N/P-dependence of transfection activity and the morphology of the lipoplexes as revealed by AFM. Morphological study with AFM provided useful information for understanding the basis of lipoplexes with superior activity and for design strategies leading to optimally efficient gene carriers. Our recent efforts have demonstrated that such a DCP-polyaimie-based liposomes

The authors thank Dr. Robert C. MacDonald for helpful comments and discussion. The present work was supported by "Grant-in-Aid for Scientific Research on Innovative Areas 2006 (Molecular Science of Fluctuations towards Biological Functions)" (201107510, 23107713) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)

Anderson, D. G., Lynn, D. M. & Lange, R. (2003) Semi-automated synthesis and screening of

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coating with glycocluster nanoparticles. *J. Am. Chem. Soc.* 125, 3455–3457. Asai, T., Matsushita, S., Kenjo, E., Tsuzuku, T., Yonenaga, N., Koide, H., Hatanaka, K.,

Behr, J.-P., Demeneix, B., Loeffler, J. P. & Perez-Mutul, J. (1989) Efficient gene transfer into

Bell, P. C., Bergsma, M., Dolbnya, I. P., Bras, W., Stuart, M. C. A., Rowan, A. E., Feiters, M. C.

Boussif, O., Lezoulch, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B. & Behr,

a large library of degradable cationic polymers for gene delivery. *Angew. Chem. Int.* 

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Dewa, T., Nango, M., Maeda, N., Kikuchi, H. & Oku, N. (2011). Dicetyl Phosphate-Tetraethylenepentamine-Based Liposomes for Systemic siRNA Delivery.

mammalian primary endocrine cells with lipopolyamine-coated DNA. *Proc. Natl.* 

& Engberts, J. B. F. N. (2003) Transfection mediated by gemini surfactants: Engineered escape from the endosomal compartment*. J. Am. Chem. Soc.* 125, 1551–

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Scheme 2. Synthetic strategy for peptide-phospholipid conjugates

(SPDP) to form compound **4**, followed by coupling with oligopeptides, (Arg)*n*Cys (R*n*C; *n* = 5, 8). Undetachable type of analogous conjugates (DPPE-S-R*n*) were also synthesized with a coupling agent, *N*-(4-maleimidobutyroxy)succinimide (GMBS) and DPPE to form compound **5**, followed by coupling with (Arg)nCys. When the disulfide-type conjugates were applied to gene delivery assay in vitro, 3-5 times higher trasnfection activity was observed compared with the corresponding undetachable conjugates. This result suggests that such a cleavable type of peptide-phospholipid conjugate is one of promising components to assemble effective non-viral vectors.
