Abstract

Cationic dendrimers are a potential nonviral vector for gene therapy because of their ability to form electrostatic dendriplexes with DNA as well as their lack of immunogenicity. The structure of the dendriplexes can influence their interactions with cells and hence the transfection efficiency; however, the supramolecular structure of DNA-dendrimer complexes formed under different conditions and its impact on cellular uptake and gene transfection efficiency remain to be explored in detail. Using small angle X-ray scattering, here we show that DNA in the complexes with poly(amidoamine) (PAMAM) G4 dendrimer exhibited four distinct packaging states modulated by the charge density of the dendrimer prescribed by its average degree of protonation (dp, the average number fraction of protonated amine groups in dendrimer). However, we also discuss the effect of charge ratio of the dendriplexes. On this side, we find that decreasing the charge ratio of the dendriplexes affects as increasing the charge density of dendrimer, which means the four distinct packing structures also show up when varying the charge ratio of the dendriplexes. In addition, we also discussed the isotope effect of the dendriplexes. Overall, the various the charge density and charge ratio indeed alter the conformation of the dendriplexes, and we also build a the morphological map of the dendriplexes along two coordinates: charge density of dendrimer” and the “charge ratio” for understanding the effect of these two variations clearly.

Table of Contents
Abstract i
Acknowledgement ii
Table of Contents iv
List of Illustrations v
Chapter 1 Introduction 1
1.1 Background 1
1.2 Literature Review 2
1.2.1 Introduction of the Poly(amidoamine) (PAMAM) Dendrimers 2
1.2.2 Bio-applications of dendrimer 8
1.2.3 Introduction of Deoxyribonucleic acid (DNA) 11
1.2.4 The beads-on-a-string structure of chromatin 15
1.2.5 The Self-Assembly Behavior of DNA-Dendrimers Complexes 22
1.2.6 Computer simulations of the complex of DNA-like PE with dendrimer 29
Chapter 2 Experiment Section 31
2.1 Materials 31
2.2 Preparation of the DNA-PAMAM G4 dendrimer complexes 31
2.3 SAXS experiment 32
Chapter 3 Results and Discussion 33
3.1 Four distinct structure of dendriplex 33
3.1.1 The square columnar phase 36
3.1.2 The beads-on-a-string structure 39
3.1.3 The hexagonally-packed superhelices structure 50
3.1.4 The coexistence of square columnar phase and hexagonally-packed DNA superhelix 57
3.2 The morphological map of DNA-PAMAM G4 complexes 60
3.2.1 The effect of dendrimer charge density 60
3.2.2 The effect of charge ratio (N/P ratio) 66
3.2.3 The morphological map 73
3.3 The isotope effect 75
Chapter 4 Conclusions 77
Reference 79

Reference
[1] Kabanov, V. A.; Sergeyev, V. G.; PyshKina, O. A.; Zinchenko, A. A.; zezin, A.B.; Joosten, J. G. H.; Brackman, J.; Yoshikawa, K., Macromolecules, 2000, 33, 9587
[2] Melnikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc., 1995, 117, 9951
[3] Plum, G. E.; Arscott, P. G.; Bloomfield, V. A. Biopolymers, 1990, 30, 631
[4] Dias, R. S.; Pais, A.; Miguel, M.G.; Lindman, B. Colloids Surf. A., 2004, 250, 115
[5] Chen, W.; Turro, N. J.; Tomalia, D. A. Langmuir 2000, 16, 15
[6] Zheng M,; Jagota, A.; Semke E. D.;Diner, B. A.; Mclean, R. S.; Lustic, S. R.; Richardson, R. E.;Tassi, N. G., Nat. Matter., 2003, 2, 338
[7] Dufes, C.; Uchegbu, I. F.; Schatzlien, A. G., Adv. Drug. Delivery. Rev., 2005, 57, 2177
[8] Fant, K.; Esbjorner, E. K.; Lincoln,P.; Norden,B., Biochemistry, 2008, 47, 1732
[9] Newkome, G. R. Advances in Dendritic Macromolecules, JAI Press: Greenwich, CT, 1993
[10] KukowskaLatallo J. F.; Bielinska A. U.; Johnson J.; Spindler R.; Tomalia D. A.; Baker J. R., Proc. Nati. Acad. Sci., 1996, 93, 4897
[11] Tomalia, D.A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J., Tokyo, 1985, 17, 117
[12] Newkome, G. R.; Yao, Z. Q.; Baker, G. R.; Gupta, V. K. J. Org. Chem., 1985, 50, 2003
[13] Fréchet, J. M. J.; Tomalia, D.A. “Dendrimers and Other Dendritic polymers”, John Wiley& Sons, Ltd, 2001, 23
[14] Tomalia, D.A. Chemistry Today, 2005, 23, 41 DOI: 10.1002/anie.199001381
[15] Naylor, A. M.; Goodard III, W. A.; J. Am. Chem. Soc., 1989, 111,2339
[16] Tomalia,D.A.; Naylor, A. M.; Goodard III, W. A. Angew. Chem., Int. Ed. Engl. 1990, 29, 138
[17] Higgins, J. S.; Benĩot, H. C. Plymers and Neutron Scattering Press, Oxford, 1994
[18] Prosa, T, J.; Bauer, B. J.; Amis, E. J. Macromolecules, 2001, 34, 4897
[19] Cakara,D.; Kleimann, J.; Borlovec, M., Macromolecule, 2001, 36, 4201
[20] Van Duijvenbode, R.C.; Borkovec, M.; Koper, G. J.M.; Polymer, 1998, 39, 2657
[21] Brothers II, H. M.; Piehler, L. T.; Tomalia, D. A. J.Chromatogr A., 1998, 814, 233
[22] Caminade, A. M.; Padié, C.; Laurent, R.; Maraval, A.; Majoral, J. P., Sensors. 2006, 6, 901
[23] Andreasson, B.; Nordenskiöld, L.; Schultz, J., Biophys. J., 1996, 70, 2847
[24] Singh, P., Bioconjugate Chem., 1998, 9, 54
[25] Liu, M.; Fréchet, J. M. J., Pharmaceut. Sci, Technol. Today, 1999, 2, 393
[26] Zeng, F.; Zimmerman, S. C., Chem. Rev., 1997, 97, 1681
[27] Bieniarz, C., Dendrimers: Applications to pharmaceutical and Medicinal Chemistry, 1998, 18, 55
[28] Alivisatos, P. A.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J., Nature, 1996, 382, 609
[29] Mirkin,C.A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J., Nature, 1996, 382, 607
[30] Braun,E.; Eichen, Y.; Sivan, U.;Ben-Yoseph, G., Nature, 1998, 391, 775
[31] Mao, C.; Sun, W.; Seeman, N.C., J. Am. Chem. Soc., 1999, 121, 5437
[32] Anatoly, A. Z.; Ning, C., J. Phys.: Condens. Matter, 2006, 18, R453
[33] Chung, Y. B.; Nardone, C.; Hinkle, D. C., J. Mol. Biol., 1990, 216, 939
[34] Morita, M.; Tasaka, M.; Fujisawa, H., Virology, 1993, 193, 748
[35] Shibata, H.; Fujisawa, H.; Minagawa, T., J. Mol. Biol., 1987, 196, 845
[36] Cerritelli, M. E.; Cheng, N. Q.; Rosenberg, A. H.; McPherson, C. E.; Booy, F. P.; Steven, A. C., Cell, 1997, 91, 271
[37] Marieb, E. N.; Mallatt, J.; Human Anatomy, chapter2, 43, third edition.
[38] Olins, A. L.; Olins, D. E., Since, 1974, 183, 330
[39] Kornberg, R. D., Science, 1974, 184, 868
[40] Finch, J. T.; Lutter, L. C.; Rhodes, D.; Brown, R. S.; Rushton, B.; Levitt, M.; Klug, A., Nature, 1977, 269, 29
[41] Leuba, S. H.; Yang, G.; Roberti, C.; Samori, B.; vanHoldeo, K., Proc. Nati. Acad. Sci., 1994, 91, 11621
[42] Jeng, U. S.;Lin, T. L.; Lin, J. M.; Ho, D. L., Phys. B., 2006, 386, 865
[43] Baldwin, J. P.; Boseley, P. G.; Bradbury, E.M., Nature,1975, 253, 245
[44] Bradbury, E. M., BioEssays, 1992, 14, 9
[45] Lagar, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J., Nature, 1997, 389, 251
[46] Welch. P. et al., Macromolucules, 2000,33,6159.
[47] Sakaue, T.; Löwen, H., Phys. Rev. E., 2004, 70, 021801
[48] Larin. S.V. et al., J. Phys. Chem. B, 2010,114,2910
[49] Maiti. P. K. et al., Nano Letters., 2008,6,2478
[50] D. R. Lide Ed., CRC Handbook of chemistry and physics, 80th Ed. (CRC Press, Boca Raton, 1999). Some data were obtained from the 57th Ed, R. C. Weast (1976)
[51] Evans,H. M.; Ahmad, A.; Ewert, K.;Pfohl, T.; Martin-Herranz, A.; Briunsma, R. F.; Safinya, C. R., Phys. Rev. Lett., 2003, 91, 7, 075501-1
[52] Liu, Y. C.; Chen, H. L.; Su, C. J.; Liu, H. K.; Liu, W. L.; Jeng, U., Macromolecules, 2005, 38, 9493
[53]Su, C. J., Biomarcomolecule,
[54] Wallin, T.; Linse, P., J. Phys. Chem., 1996, 100, 17873
[55] Jonsson, M.; Linse, P., J. Phys. Chem., 2001, 115, 10975
[56] Kunze, K. K.; Netz, R. R., Phys.Rev. Lett., 2000,85, 4389
[57] Kunze, K. K.; Netz, R. R., Phys.Rev. E., 2002, 66, 011918
[58] Zinchenko, A. A.; Yoshikawa, K.; Baigl, D., Phys.Rev. Lett., 2005, 95, 228101
[59] D. R. Lide Ed., CRC Handbook of chemistry and physics, 80th Ed. (CRC Press, Boca Raton, 1999). Some data were obtained from the 57th Ed, R. C. Weast (1976)
[60] F. Franks, Water: 2nd Edition A matrix of life, (Royal Society of Chemistry, Cambridge, 2000)