Dendrimer Synthesis and Applications: Branching out into Biology

1. Dendrimer Synthesis and Applications: Branching out into Biology Organic Chemistry Seminar April 28th, 2005 William Pomerantz Gellman Group

2. 2 Dendritic: Nature’s Architecture Dendrimer- Greek roots: dendra- tree, mer- segment mm Neuron m Tree nm Dendrimer Tomalia, D., A.; Frechet, J. M. J. J. Polym. Sci., Part A: Polym. Chem.2002, 40, 2719-2728 http://inside.salve.edu/walsh/

3. Globular Dendrimers as Biomimetics Molecular Modeling of Dendrimer Structure 16 Å 67 Å Hemoglobin Prealbumin Insulin Cytochrome C Maiti, P. K.; Tahir, C.; Wang, G.; Goddard, W. A. I. Macromolecules2004, 37, 6236 Tomalia, D., A.; Frechet, J. M. J. J. Polym. Sci., Part A: Polym. Chem.2002, 40, 2719-2728

4. Dendrimer 101 Generation (GX)Defines the level of branching within the dendrimer shell. At high Generations dendrimers become spherical G2 G1 G0 LoadingReactive/diagnostic groups can be attached to to the surface of the dendrimers efficiently and with a predictable display

5. Generation Growth GX = generation G0 Core Monomer

6. Generation Growth GX = generation G1 Core Monomer

7. Generation Growth GX = generation G2 Core Monomer

8. Generation Growth GX = generation G3 Core Monomer

9. Dendrimer 101 Generation (GX)Defines the level of branching within the dendrimer shell. At high Generations dendrimers become spherical G2 G1 G0 LoadingReactive/diagnostic groups can be attached to to the surface of the dendrimers efficiently and with a predictable display

10. Dendrimer 101 Polydispersity Index (PDI) Indication of the distribution of molecular weights within a sample. PDI=1 is monodisperse PDI= Mw/Mn Size Exclusion Chromatography G3 G2 G1 G4 Intensity Time (Minutes) de Brabander-van den Berg, E., M. M.; Meijer, E. W. Angew. Chem. Int. Ed.1993, 32, 1308

11. 11 Outline Gene Delivery Cancer Therapy Encapsulation Synthesis Divergent Convergent Applications Multivalency MRI Conclusions and Outlook

12. 12 Synthetic Considerations Polydispersity (PDI) Reagents Cheap High Reactivity Easily Removable Very Narrow PDI: PDI= Mw/Mn Reactions High Yielding Minimal side reactions Purify intermediates

13. 13 Divergent Dendrimer Synthesis G0 G1 G2 Multivalent Monomer G3 Dendrimer Core

14. 14 PAMAM Dendrimer Synthesis (Divergent) G0 .. G1 G0.5 Very Narrow PDI: PDI= Mw/Mn Tomalia, D., A. and co-workers. Macromolecules1986, 19, 2466, Polym. J. (Tokyo)1985, 17, 117

15. 15 Polydispersity vs. Dendrimer Purity Each reaction assumes 99.5% conversion Hummeln, J. C.; van Dongen, J. L. J.; Meijer, E. W. Chem. Eur. J.1997, 3, 1489 Kallos, G.; et al. J.Rapid Commun. Mass Spectrom1991, 5, 383

16. 16 Polydispersity vs. Dendrimer Purity Each reaction assumes 99.5% conversion Hummeln, J. C.; van Dongen, J. L. J.; Meijer, E. W. Chem. Eur. J.1997, 3, 1489 Kallos, G.; et al. J.Rapid Commun. Mass Spectrom1991, 5, 383

17. Convergent Synthesis Dendrimer Core G3 Multivalent Monomer Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc1990, 112, 7638-7647

18. 18 Dendrimer Wedge Synthesis (Convergent) G0 G0 G1 G2 Frechet and co-workers; J. Am. Chem. Soc.1990, 112, 7638 J. Control. Release2000, 65, 121

19. 19 G2 Dendrimer Synthesis (Convergent) G2-Dendrimer G2 G2-Br Frechet and co-workers; J. Am. Chem. Soc.1990, 112, 7638 J. Control. Release2000, 65, 121

20. 20 G2 Dendrimer Synthesis (Convergent) R = Bn R = H R = (CH2CH2O)16CH3 G2-Dendrimer Frechet and co-workers; J. Am. Chem. Soc.1990, 112, 7638 J. Control. Release2000, 65, 121

21. 21 Synthetic Comparison Convergent Divergent Advantages: Fewer simultaneous reactions Standard purification Intermediates characterizable Differentiation Monodisperse Rapid synthesis Cheap reagents Exponential growth Large dendrimers attainable Disadvantages: Multiple side reactions (intra/inter) Large excess of reagents Low polydispersity Slower growth process Mid-sized dendrimers

22. 22 Outline Gene Delivery Cancer Therapy Encapsulation Synthesis Divergent Convergent Applications Multivalency MRI Conclusions and Outlook

23. 23 Molecular Encapsulation Surface Interbranch Core Guests = Space-Filling Model Jansen, J. F. G. A.; de Brabander-van den Berg, E., M. M.; Meijer, E. W. Science1994, 266, 1226

24. 24 Molecular Encapsulation Surface Interbranch Core Guests = Space-Filling Model Jansen, J. F. G. A.; de Brabander-van den Berg, E., M. M.; Meijer, E. W. Science1994, 266, 1226

25. 25 Molecular Encapsulation-“Dendritic-Box” Lid BOX PPI Dendrimer Jansen, J. F. G. A.; et al. Science1994, 266, 1226 de Brabander-van den Berg, et al. Angew. Chem. Int. Ed.1993, 32, 1308

26. 26 Molecular Encapsulation-“Dendritic-Box” PPI Dendrimer Jansen, J. F. G. A.; et al. Science1994, 266, 1226 de Brabander-van den Berg, et al. Angew. Chem. Int. Ed.1993, 32, 1308

27. 27 “Phe-Box” Probe Encapsulation 1) Probe + Coupling 1) Coupling 2) Probe 1) Probe + Coupling 2) 12M HCl ESR Probe Jansen, J. F. G. A.; et al. Science1994, 266, 1226

28. 28 Unimolecular Micelle Containers G1 G2 G3 “Frechet” Polyaryl- ether dendrimer G3 [Pyrene]water 10-6 M G2 G1 [Dendrimer] Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release2000, 65, 121

29. 29 Drug Encapsulation and Release Dialysis membrane Indomethacin Water bath Free Drug % Release Drug + G3 pH =7 37oC Time (hr) Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release2000, 65, 121

30. 30 Outline Gene Delivery Cancer Therapy Encapsulation Synthesis Divergent Convergent Applications Multivalency MRI Conclusions and Outlook

31. 31 Gene Delivery DNA transported to nucleus via viral or synthetic molecules Cell Gene Product www.comet.itrcindia.org http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

32. 32 Key Processes for Gene Delivery Bind DNA 1. Polycation Endosomal Release Transfection Cellular Uptake 3. 2. Boussif, O.; et al. Proc. Natl. Acad. Sci. USA1995, 92, 7297. Behr, J. P. Acc. Chem. Res.1993, 26, 274

33. 33 Dendrimer Transfection Efficiency pLys115 Gn +/- Charge ratio Haenzler, J.; Szoka, F. C. J. Bioconjugate Chem.1993, 4, 372 Tang, M. X.; Redemann, C. T.; Szoka, F. C. J. Bioconjugate Chem.1996, 7, 703

34. 34 Dendrimer Transfection Efficiency pLys115 Superfect Gn +/- Charge ratio Haenzler, J.; Szoka, F. C. J. Bioconjugate Chem.1993, 4, 372 Tang, M. X.; Redemann, C. T.; Szoka, F. C. J. Bioconjugate Chem.1996, 7, 703

35. 35 Transfection/Cytotoxicity Comparison Luciferase (ng/mg) PEI 25K Branched PEI 50K Linear PEI 22K Linear PEI/ Pluronic Copolymer Cell Viability Gebhart, C. L.; Kabanov, A. V. J. Control. Release2001, 73, 401

36. 36 Increasing Transfection vs. Toxicity Phe(64)-G4 Lipofectamine Superfect Phe PAMAM Defect PAMAM Kono, K.; et al. Bioconjugate Chem.2005, 16, 208, Malik, N.; et al. J. Control. Release2000, 65, 133

37. 37 Outline Gene Delivery Cancer Therapy Encapsulation Synthesis Divergent Convergent Applications Multivalency MRI Conclusions and Outlook

38. 38 (Macromolecular) Drug Biocompatibility Drug Drug Criteria: • Water Soluble • Low Cytotoxicity • Biodistribution • tissue/cell specificity • Bioavailability • half-life in body, degradable • Reproducible Pharmacokinetics Drug Drug Drug Drug HPMA Co-polymer Duncan, and co-workers S. Hum Exp Toxicol1998, 17, 93, Eur. J. Cancer1995, 5, 766

39. 39 Enhanced Permeability and Retention (EPR) Passive Targeting of Tumor Cells Duncan, R. Nat Rev Drug Discov2003, 2, 347-360, Matsumara, Y.; et al. Cancer Res.1986, 6, 6387

40. 40 Dendrimer Design Features = Drug High MW Dendrimer High MW Dendrimer Modular Approach Frechet, J. M. J. and co-workers. Bioconjugate Chem.2002, 13, 443, Macromolecules1998, 31, 4061, Bioconjugate Chem.2002, 13, 453

41. 41 Evaluating Dendrimer Size for EPR I = G4 Dendrimer 4 kDa II G4 Dendrimer 12 kDa = Core III G2 Dendrimer 24 kDa De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem.2002, 13, 453

42. 42 Conjugation of Doxorubicin (DOX) = DOX De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem.2002, 13, 453

43. 43 Dendrimer Cytotoxicity 4 kDa % Cell Viability 12 kDa 24 kDa [Dendrimer] (mg/mL) Half-Lives of I-III all < 10 Min. De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem.2002, 13, 453

44. 44 Drug Release Studies = DOX De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem.2002, 13, 453

45. 45 Higher MW “Bow-Tie” Dendrimers G2-5 kDa G2-10 kDa G2-20 kDa G3-5 kDa G3-10 kDa G3-20 kDa G1-10 kDa G1-20 kDa Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137

46. 46 Higher MW “Bow-Tie” Dendrimers G2-5 kDa G2-10 kDa G2-20 kDa G3-5 kDa G3-10 kDa G3-20 kDa G1-10 kDa G1-20 kDa Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137

47. 47 Biodistribution and Bioavailibility In Vivo Plasma Half-Life (hrs) G1-10 kDa 8 +/- 1 G2-10 kDa 26 +/- 6 G3-10 kDa 40 +/- 4 G3-20 kDa 50 +/- 10 G3-10 kDa G3-20 kDa % Dose /g tissue Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137

48. 48 Outline Gene Delivery Cancer Therapy Encapsulation Synthesis Divergent Convergent Applications Multivalency MRI Conclusions and Outlook

49. 49 Multivalent Glycoreceptors **Binding sites shallow, Ka Monomer ~ 10-3 M Bertozzi, C. R.; Kiessling, L. L. Science2001, 291, 2357. Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555. Lee, Y. C.; Lee, R. T. Acc. Chem. Res.1995, 28, 321

50. 50 Dendrimers for Multivalent Display Size control can affect mechanism of binding Dendrimer amenable to modular design G2-PAMAM = Sugar Lundquist, J. J.; Toone, E. J. Chem. Rev.2002, 102, 555. Kanai, M.; et al. J. Am. Chem. Soc.1997, 119, 9931.