Dokument

Summary:
In this thesis, biodegradable non-viral polymeric nucleic acids delivery vectors were characterized concerning biophysicochemical parameters. In the first part of this research, to answer the questions: why the principle of DNA transfection cannot be directly applied for siRNA transfection, we investigated the complexation and aggregation mechanism of nucleic acids/polycations on the atomic and molecular scale. The MD and ITC data showed us the different nature and the different hierarchical mechanism related polycation-siRNA and polycation-pDNA complexes. All our results emphasized one point: lower N/P-ratios are especially effective for polycationic nanocarrier-based siRNA delivery, because siRNA aggregation results in a more uniform and stable complex formation at low N/P ratios already, which lead to increased siRNA delivery efficiency. This could have broad implications for the delivery of siRNA as less toxic and yet efficient delivery systems have been the bottle-neck for the translation of this promising approach into the clinical arena. In chapter 3, novel biodegradable amphiphilic copolymers hy-PEI-g-PCL-b-PEG were prepared by grafting PCL-b-PEG chains onto hyper-branched poly(ethylene imine) as non-viral gene delivery vectors. With the question: how can the graft densities of PCL-b-PEG chains influence the in vitro DNA delivery efficiency, our study began with the characterization of physico-chemical properties and expected that with the introducing of the grafted PCL-b-PEG chains, the in vitro DNA delivery efficiency with the grafted PCL-b-PEG chains could be improved. Of all the experimental results, buffer-capacity has almost exactly the same tendency as transfection efficiency. We assume that in all processes of DNA transfection, the endosomal escape has a really important and rate-limiting role. This opens new perspectives to advance the rational design of new gene delivery systems. The further investigation of these biodegradable grafted amphiphilic copolymers hy-PEI-g-(PCL-b-PEG)n as potential siRNA delivery vectors was showed in chapter 4. The purpose in this section was to enhance the in vivo blood circulation time and siRNA delivery efficiency of biodegradable copolymers polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hyPEI-g-PCL-b-PEG) by introducing high graft densities of PCL-PEG chains. Our study indicated that the effect of PEG on prolonged circulating depends not only on its content in a copolymer (length or percentage), but also on the structure or the shape of the amphiphilic copolymer. We demonstrated that polymeric micelles, which are formed with amphiphilic block polymers have advantages especially for in vivo siRNA delivery, and that the graft density of the amphiphilic chains can enhance the blood circulation, which is a key parameter to promote the development of safe and efficient non-viral polymeric siRNA delivery in vivo. Although the copolymers hy-PEI-g-(PCL-b-PEG)n showed positive results as pDNA and siRNA delivery vectors in chapter 3 and 4, the delivery of gene materials with these non-targeted copolymers is achieved mainly passively by the passive targeting. Therefore, to optimize these polymeric gene delivery vectors with targeting function, in chapter 5, folate conjugated PEI-g-PCL-b-PEG was examined for targeted gene delivery. Lower cytotoxicity was observed for PEI-g-PCL-b-PEG-Fol than PEI-g-PCL-b-PEG and the cellular uptake of polyplexes was enhanced by PEI-g-PCL-b-PEG-Fol in FR over-expressing KB cells compared with those by PEI-g-PCL-b-PEG. Importantly, this enhancement was inhibited by free folic acid, while did not appear in FR-negative A549 cells. All these suggested the specific cell uptake of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes via folate receptor-mediated endocytosis. Consequently, PEI-g-PCL-b-PEG-Fol/pDNA polyplexes revealed higher transfection than PEI-g-PCL-b-PEG/pDNA. Additional studies on gene transfection in vivo and utilizing these described folate-conjugated copolymers for targeted siRNA delivery are in proceeding. In Chapter 6, the novel siRNA delivery systems based on hyperflexible generation 2-4 triazine dendrimers was identified by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with their thermodynamic interaction features simulated by molecular modeling and the influence of dendrimer flexibility has systematically been investigated and discussed. In this study, molecular modeling helped to understand experimental parameters based on the dendrimers’ structural properties and molecular imaging non-invasively predicted the in vivo fate of the complexes, both techniques can efficiently support the rapid development of safe and efficient siRNA formulations that are stable in vivo.

Bibliographie / References

1. Caldwell, P.A. Kollman, A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules, Journal of the American Chemical Society, 117 (1995) 5179-5197.
2. V.K. Sharma, M. Thomas, A.M. Klibanov, Mechanistic studies on aggregation of polyethylenimine-DNA complexes and its prevention, Biotechnol Bioeng, 90 (2005) 614-620.
3. H. Kang, R. DeLong, M.H. Fisher, R.L. Juliano, Tat-conjugated PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides, Pharmaceutical Research, 22 (2005) 2099-2106.
4. V.A. Izumrudov, T.K. Bronich, M.B. Novikova, A.B. Zezin, V.A. Kabanov, Substitution reactions in ternary systems of macromolecules, Polymer Science U.S.S.R., 24 (1982) 367-378.
5. K. Jorgensen, M.J. Rice, Morphology of a very extensible insect muscle, Tissue Cell, 15 (1983) 639-644.
6. A.P. Perez, E.L. Romero, M.J. Morilla, Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents, Int J Pharm, 380 (2009) 189-200.
7. Endres, T.; Zheng, M.; Beck-Broichsitter, M.; Kissel, T., Lyophilised ready-to-use formulations of PEG-PCL-PEI nano-carriers for siRNA delivery. Int J Pharm 2012, 428 (1-2), 121-4.
8. Y. Inoue, R. Kurihara, A. Tsuchida, M. Hasegawa, T. Nagashima, T. Mori, T. Niidome, Y. Katayama, O. Okitsu, Efficient delivery of siRNA using dendritic poly(L-lysine) for loss-of-function analysis, J Control Release, 126 (2008) 59-66.
9. M. Merkel, D. Librizzi, A. Pfestroff, T. Schurrat, K. Buyens, N.N. Sanders, S.C. De Smedt, M. Behe, T. Kissel, Stability of siRNA polyplexes from poly(ethylenimine) and poly(ethylenimine)-g-poly(ethylene glycol) under in vivo conditions: effects on pharmacokinetics and biodistribution measured by Fluorescence Fluctuation Spectroscopy and Single Photon Emission Computed Tomography (SPECT) imaging, J Control Release, 138 (2009) 148-159.
10. I.D. Kim, C.M. Lim, J.B. Kim, H.Y. Nam, K. Nam, S.W. Kim, J.S. Park, J.K. Lee, Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain, J Control Release, 142 (2010) 422-430.
11. Merkel, O. M.; Zheng, M. Y.; Mintzer, M. A.; Pavan, G. M.; Librizzi, D.; Maly, M.; Hoffken, H.; Danani, A.; Simanek, E. E.; Kissel, T., Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems. Journal of Controlled Release 2011, 153 (1), 23-33.
12. Q. Yuan, E. Lee, W.A. Yeudall, H. Yang, Dendrimer-triglycine-EGF nanoparticles for tumor imaging and targeted nucleic acid and drug delivery, Oral Oncol, 46 (2010) 698-704.
13. Merkel, O. M.; Zheng, M.; Debus, H.; Kissel, T., Pulmonary gene delivery using polymeric nonviral vectors. Bioconjug Chem 2011, 23 (1), 3-20.
14. M. Merkel, D. Librizzi, A. Pfestroff, T. Schurrat, M. Behe, T. Kissel, In vivo SPECT and real-time gamma camera imaging of biodistribution and pharmacokinetics of siRNA delivery using an optimized radiolabeling and purification procedure, Bioconjug Chem, 20 (2009) 174-182.
15. M. Merkel, M.A. Mintzer, J. Sitterberg, U. Bakowsky, E.E. Simanek, T. Kissel, Triazine dendrimers as nonviral gene delivery systems: effects of molecular structure on biological activity, Bioconjug Chem, 20 (2009) 1799-1806.
16. Zhang, Y.; Zheng, M.; Kissel, T.; Agarwal, S., Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection. Biomacromolecules 2012, 13 (2), 313-22.
17. M.L. Patil, M. Zhang, O. Taratula, O.B. Garbuzenko, H. He, T. Minko, Internally cationic polyamidoamine PAMAM-OH dendrimers for siRNA delivery: effect of the degree of quaternization and cancer targeting, Biomacromolecules, 10 (2009) 258-266.
18. M. Pavan, A. Danani, S. Pricl, D.K. Smith, Modeling the multivalent recognition between dendritic molecules and DNA: understanding how ligand "sacrifice" and screening can enhance binding, J Am Chem Soc, 131 (2009) 9686-9694.
19. T. Ehtezazi, U. Rungsardthong, S. Stolnik, Thermodynamic analysis of polycation-DNA interaction applying titration microcalorimetry, Langmuir, 19 (2003) 9387-9394.
20. Zheng, M.; Pavan, G. M.; Neeb, M.; Schaper, K., A.; Danani, A.; Klebe, G.; Merkel, M. O.; Kissel, T.: Targeting the blind spot of polycationic nanocarrier-based siRNA delivery (Submitted to ACS Nano, 2012)
21. A. Agrawal, D.H. Min, N. Singh, H. Zhu, A. Birjiniuk, G. von Maltzahn, T.J. Harris, D. Xing, S.D. Woolfenden, P.A. Sharp, A. Charest, S. Bhatia, Functional delivery of siRNA in mice using dendriworms, ACS Nano, 3 (2009) 2495-2504.
22. C. Shen, J. Zhou, X. Liu, J. Wu, F. Qu, Z.L. Zhang, D.W. Pang, G. Quelever, C.C. Zhang, L. Peng, Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer complexes, Org Biomol Chem, 5 (2007) 3674-3681.
23. L. Juliano, Intracellular delivery of oligonucleotide conjugates and dendrimer complexes, Ann N Y Acad Sci, 1082 (2006) 18-26.
24. T. Tsutsumi, H. Arima, F. Hirayama, K. Uekama, Potential Use of Dendrimer/α-Cyclodextrin Conjugate as a Novel Carrier for Small Interfering RNA (siRNA), Journal of Inclusion Phenomena and Macrocyclic Chemistry, 56 (2006) 81-84.
25. Abschnitt der Pharmazeutischen Prüfung Sep. 2010
26. In Kapitel 7 wurden neuartige bioabbaubare und biokompatible Polymere der Zusamensetzung Poly (PEG-co-(BMDO-co-DMAEMA) für die Gen-Transfektion erfolgreich bezüglich ihrer physikalisch-chemischen und in vitro biologischen Eigenschaften charakterisiert. Die quarternierten Copolymere zeigten niedrige Zytotoxizitä t als die quarternisierten Copolymere sowie positive Ergebnisse in der DNA-Transfektion in vitro.
27. Geburtsort: Beijing, China Staatsangehörigkeit: Chinesisch Anschrift: Wehrdaer Weg 3, 35037 Marburg E-Mail: mengyao_zheng@hotmail.com Ausbildung und Berufspraxis Jul. 2001
28. Curriculum Vitae Persönliche Daten Name: Mengyao Zheng Geburtsdatum: 17.02.1983
29. In Kapitel 6 wurden hyperflexible Triazin-Dendrimere der Generationen 2-4 als neuartige siRNA-Delivery-Systeme bezüglich ihrer physikalisch-chemischer und biologischer in vitro und in vivo Eigenschaften untersucht und diskutiert. In diesem Kapitel wurden die thermodynamische Eigenschaften durch molekulare Modellierung simuliert, und der Einfluss der Flexibilitä t wurde systematisch untersucht und diskutiert.
30. PEI-g-PCL-b-PEG-Fol/pDNA Polyplexe über Folat-Rezeptor-vermittelte Endozytose. Die in vitro DNA Transfektionseffizienz wurde deutlich erhöht durch die Kupplung von Folsä ure. Die zusä tzlichen Untersuchungen in vivo und Nutzung dieser beschriebenen Folat-konjugierten Copolymeren für gezielte siRNA Transfektion sind in Bearbeitung.
31. I. Mellman, Endocytosis and molecular sorting, Annu Rev Cell Dev Biol, 12 (1996) 575-625.
32. Zheng, M.; Merkel, M. O.; Librizzi, D.; Kilic, A.; Liu, Y.; Renz, H.; Kissel, T.: Enhancing in vivo long-circulating and siRNA delivery efficiency of biodegradable high grafted hy-PEI-g-PCL-b-PEG copolymers (Accepted by Biomaterials, 2012)
33. Mengyao Zheng, Li Liu, Damiano Librizzi, Thomas Renette, Olivia M. Merkel, Thomas Kissel: Folate conjugated copolymer (Fol-PEG-PCL-hyPEI) for efficient and targeted in vitro and in vivo sirna delivery, Controlled Release Society Local Chapter Meeting Germany, Wuerzburg, Germany, March 29-30, 2012
34. Dendron-DNA Interactions: Insights into Multivalency through a Combined Experimental and Theoretical Approach, Chemistry, 16 (2010) 4519-4532.
35. Wahlpflichtpraktikum, Institut für Pharmazeutische Chemie, Philipps-Universitä t, Marburg: Isolierung und Charakterisierung rekombinanter RNA-und DNA-Polymerasen Oct. 2009
36. Banaszak Holl, Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability, Bioconjug Chem, 17 (2006) 728-734.
37. J. Suh, H.J. Paik, B.K. Hwang, Ionization of Poly(ethylenimine) and Poly(allylamine) at Various pH's, Bioorganic Chemistry, 22 (1994) 318-327.
38. In Kapitel 3-5 werden die Copolymer hy-PEI-g-(PCL-b-PEG)n als neuartige Transfektionpolymere beschrieben. In dieser Untersuchung wurden nur Copolymere mit kurzen PCL-Segmenten
39. In Kapitel 3 haben wir anhand der Untersuchung des Einflusses der Kupplungsdichten von PCL-b-PEG-Ketten auf physikalisch-chemische Eigenschaften, DNA-Komplexierung und Transfektionseffizienz gefunden, dass der " endosomal escape " eine sehr wichtige Rolle für die
40. Liu, L.; Zheng, M.; Renette, T.; Kissel, T., Modular Synthesis of Folate Conjugated Ternary Copolymers: Polyethylenimine-graft-Polycaprolactone-block-Poly(ethylene glycol)-Folate for Targeted Gene Delivery. Bioconjug Chem 2012. (contributed equally first authors)
41. Li Liu, Mengyao Zheng, Markus Benfer, Thomas Kissel: Multifunctional nano carrier based on PEI-g-PCL-b-PEG-folate for targeted gene delivery. the 3rd European Science Foundation Summer School" Nanomedicine 2011 ", Lutherstadt Wittenberg, Germany, 19-24 June 2011
42. J. McCarroll, H. Baigude, C.S. Yang, T.M. Rana, Nanotubes functionalized with lipids and natural amino acid dendrimers: a new strategy to create nanomaterials for delivering systemic RNAi, Bioconjug Chem, 21 (2010) 56-63.
43. M. Merkel, A. Beyerle, D. Librizzi, A. Pfestroff, T.M. Behr, B. Sproat, P.J. Barth, T. Kissel, Nonviral siRNA delivery to the lung: investigation of PEG-PEI polyplexes and their in vivo performance, Mol Pharm, 6 (2009) 1246-1260.
44. Abitur, Gymnasium, Beijing, China (Note: sehr gut)
45. Endres, T.; Zheng, M.; Beck-Broichsitter, M.; Samsonova, O.; Debus, H.; Kissel, T., Optimising the self-assembly of siRNA loaded PEG-PCL-lPEI nano-carriers employing different preparation techniques. J Control Release 2012.
46. T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N [center-dot] log(N) method for Ewald sums in large systems, The Journal of Chemical Physics, 98 (1993) 10089-10092.
47. Philipps-Universitä t Marburg Fachbereich Pharmazie Feb.2009
48. A.L. Bolcato-Bellemin, M.E. Bonnet, G. Creusat, P. Erbacher, J.P. Behr, Sticky overhangs enhance siRNA-mediated gene silencing, Proc Natl Acad Sci U S A, 104 (2007) 16050-16055.
49. Mengyao Zheng, Olivia Merkel, Manuel Neeb, Michael Hellwig, Thomas Kissel: Structural Conformations and Nucleic Acid Location within Amphyphilic hy-PEI-PCL-mPEG Complexes as Non-Viral Vectors, Lecture, Controlled Release Society Local Chapter Meeting Germany, Jena, Germany, March 15-16, 2011
50. M.L. Patil, M. Zhang, S. Betigeri, O. Taratula, H. He, T. Minko, Surface-modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery, Bioconjug Chem, 19 (2008) 1396-1403.
51. M.X. Tang, F.C. Szoka, The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes, Gene Ther, 4 (1997) 823-832.
52. R. Jevprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. McKeown, A. D'Emanuele, The influence of surface modification on the cytotoxicity of PAMAM dendrimers, Int J Pharm, 252 (2003) 263-266.
53. M. Dykxhoorn, D. Palliser, J. Lieberman, The silent treatment: siRNAs as small molecule drugs, Gene Ther, 13 (2006) 541-552.
54. Olivia Merkel, Mengyao Zheng, Meredith Mintzer, Giovanni Pavan, Damiano Librizzi, Eric Simanek, Thomas Kissel: Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems, Controlled Release Society Local Chapter Meeting Germany , Jena, Germany, 15-16 March, 2011
55. Weiterentwicklungen der beschriebenen nicht-virale Gen-Transfektion-Systeme sind immer denkbar. In Kapitel 2 haben wir die Komplexierung und den Aggregationsmechanismus von Nukleinsä uren mit Polykationen auf atomarer und molekularer Ebene untersucht. In diesem Projekt wurden molekularen Modellierung, molekulardynamische Simulation, isotherme Titrationskalorimetrie und andere Charakterisierungsmethoden angewendet. Trotzdem besteht immer Bedarf für neue Untersuchungsmethode zur Charakterisierung der pDNA-oder siRNA-Lokalisierung innerhalb der Komplexe. Es ist daher sinnvoll, neue mikroskopische Methoden weiterzuentwickeln.
56. X. Liu, P. Rocchi, F.Q. Qu, S.Q. Zheng, Z.C. Liang, M. Gleave, J. Iovanna, L. Peng, PAMAM dendrimers mediate siRNA delivery to target Hsp27 and produce potent antiproliferative effects on prostate cancer cells, ChemMedChem, 4 (2009) 1302-1310.
57. Mattheis, C.; Zheng, M. Y.; Agarwal, S., Closing One of the Last Gaps in Polyionene Compositions: Alkyloxyethylammonium Ionenes as Fast-Acting Biocides. Macromolecular Bioscience 2012, 12 (3), 341-349.
58. M. Pavan, M.A. Mintzer, E.E. Simanek, O.M. Merkel, T. Kissel, A. Danani, Computational insights into the interactions between DNA and siRNA with "rigid" and "flexible" triazine dendrimers, Biomacromolecules, 11 (2010) 721-730.
59. M. Pavan, L. Albertazzi, A. Danani, Ability to adapt: different generations of PAMAM dendrimers show different behaviors in binding siRNA, J Phys Chem B, 114 (2010) 2667-2675.
60. M.A. Mintzer, O.M. Merkel, T. Kissel, E.E. Simanek, Polycationic triazine-based dendrimers: effect of peripheral groups on transfection efficiency, New J Chem, 33 (2009) 1918-1925.
61. I. Andricioaei, M. Karplus, On the calculation of entropy from covariance matrices of the atomic fluctuations, The Journal of Chemical Physics, 115 (2001) 6289-6292.
62. L. Waite, S.M. Sparks, K.E. Uhrich, C.M. Roth, Acetylation of PAMAM dendrimers for cellular delivery of siRNA, BMC Biotechnology, 9 (2009).
63. W.R. Sanhai, J.H. Sakamoto, R. Canady, M. Ferrari, Seven challenges for nanomedicine, Nat Nanotechnol, 3 (2008) 242-244.
64. M. Breunig, U. Lungwitz, R. Liebl, A. Goepferich, Breaking up the correlation between efficacy and toxicity for nonviral gene delivery, Proc Natl Acad Sci U S A, 104 (2007) 14454-14459.
65. Merkel, O. M.; Beyerle, A.; Beckmann, B. M.; Zheng, M. Y.; Hartmann, R. K.; Stoger, T.; Kissel, T. H., Polymer-related off-target effects in non-viral siRNA delivery. Biomaterials 2011, 32 (9), 2388-2398.
66. Zheng, M. Y.; Liu, Y.; Samsonova, O.; Endres, T.; Merkel, O.; Kissel, T., Amphiphilic and biodegradable hy-PEI-g-PCL-b-PEG copolymers efficiently mediate transgene expression depending on their graft density. International Journal of Pharmaceutics 2012, 427 (1), 80-87. (contributed equally first authors)
67. Svenson, Dendrimers as versatile platform in drug delivery applications, European Journal of Pharmaceutics and Biopharmaceutics, 71 (2009) 445-462.

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten