1. CALIXARENE-BASED NANOSYSTEMS FOR GENE DELIVERY Francesco Sansone, Laura Baldini, Alessandro Casnati, Gaetano Donofrio, Miroslav Dudič, Claudio Rivetti, Rocco Ungaro Dipartimento di Chimica Organica e Industriale Università di Parma Unità INSTM

2. “Gene Therapy” Delivery of nucleic acids to patients for therapeutic purposes Vector Vector-DNA complex Endosome Protein Synthesis Nucleus Therapeutic DNA Vector Vector-DNA complex Cellular membrane Gene Therapy can be used either to treat diseases caused by defective genes (e.g. Cystic Fibrosis) or to express other genes for therapeutic treatment (e.g. to express genes inside cancer cells to kill them)

3. Adenovirus / Adeno-associated Virus Retroviral Vectors Herpes Simplex Virus Lentiviral Vectors DNA Delivery Systems for Gene Therapy Genes, unlike most drugs, are degraded when introduced into the body. To protect genes, we need to develop delivery systems (Vectors). SYNTHETIC NON-VIRAL VECTORS No hazard from native gene expression Less efficient gene transfer than viruses Lower tissue specificity VIRAL VECTORS Possible infection risk (remove as many genes as possible) Immune reaction (memory from previous infections) Protect therapeutic genes well Show some specificity Liposomes Cationic Polymers Gemini Surfactants Glycocluster Nanoparticles

4. “Gene Therapy” with non-viral, synthetic vectors Felgner et al PNAS 1987 Behr et al PNAS 1989 Gao et al Biochim. Biophys. Res. Commun. 1991 Li and Huang Gene Therapy 2006

5. Guanidinium-Calix[n]arenes Tetrahedron 2004, 60, 11621-11626 JACS 2006, 128, 14528-14536 CONE conformation Conformationally MOBILE 1,3-ALTERNATE

6. Synthesis

7. Water solubility  10 -4

8. Cell Transfection 4G4Oct- cone 4G4Oct- cone 4G4Oct- cone 4G4Oct- cone GenePORTER transfection reagent 5  M 40  M20  M10  M a 4G4Oct- cone 4G4Oct- cone 4G4Oct- cone 4G4Oct- cone GenePORTER transfection reagent 5  M 40  M20  M10  M a

9. Transfection Calix[4] octyl Calix[4] hexyl without DOPE cone mobile Calix[4] propyl Calix[4] methyl Calix[4] propyl with DOPE cone 1,3-alt No Transfection Calix[6] methyl Calix[8] methyl mobile

10. Plasmid DNA 1 nM Plasmid DNA and CONE Guanidinium-Calix[4]arenes Plasmid DNA + 4G4Pr-cone (1  M) Plasmid DNA + 4G4Oct-cone (1  M) Plasmid DNA + 4G4Hex-cone (1  M) JACS 2006, 128, 14528-14536

11. Plasmid DNA + 6G6Me-mobile (1  M) Plasmid DNA + 8G8Me-mobile (1  M) Plasmid DNA + 4G4Pr-alt (1  M) Plasmid DNA + 4G4Me-mobile (1  M) Plasmid DNA and “OTHERS” Guanidinium-Calixarenes

12. Effect of ETHANOL on CONE Guanidinium-Calix[4]arenes Plasmid DNA 1 nM + 5% EtOH Plasmid DNA + 4G4Pr-cone + 5% EtOH Plasmid DNA + 4G4Hex-cone + 15% EtOH Plasmid DNA + 4G4Oct-cone + 15% EtOH

13. = Transfection !

14. Plasmid DNA + 4G4Pr-alt + 5% EtOH Plasmid DNA + 4G4Me-mobile + 5% EtOH Effect of ETHANOL on 1,3-ALTERNATE and MOBILE Guanidinium-Calix[4]arenes

15. = + DOPE Transfection !

16. Plasmid DNA + 6G6Me-mobile +5% EtOH Plasmid DNA + 8G8Me-mobile +5% EtOH Effect of ETHANOL on MOBILE Guanidinium-Calix[6]- and Calix[8]arenes

17. = No Transfection!

18. Conclusions A new series of calixarene-based ligands was synthesised able to bind plasmid DNA Some of them give cell transfection thanks to their DNA condensation capability Condensation and transfection properties are strongly dependent on conformation, structure and size of the ligands

19. Interaction with plasmid DNA Tetrahedron 2004, 60, 11621-11626 Electrophoresis Mobility Shift Assay 6G6Me-mobile 4G4Pr-cone 8G8Me-mobileDNA Ethidium Bromide Displacement Assays JACS 2006, 128, 14528-14536 Melting curves Linear Plasmid + 4G4Pr-cone + 8G8Me-mobile + 4G4Pr-alt JACS 2006, 128, 14528-14536

20. 1 H NMR spectra in D 2 O (300 MHz, 300 K)

21. Dilution experiments by 1 H NMR in D 2 O (300 MHz, 300 K) with 4G4Hex-cone 0.2 mM 0.5 mM 1.0 mM 1.6 mM 2.5 mM 5.0 mM 10.0 mM cmc 0.2 mM

22. LogD NMR DOSY experiments with 4G4Hex-cone in D 2 O (300 MHz, 300 K) Hydrodynamic radius r H of monomer: 8.9 Å Hydrodynamic radius r H of aggregate: 32.7 Å

23. Miroslav Dudič Laura Baldini, Alessandro Casnati Rocco Ungaro Dept. of Organic and Industrial Chemistry Acknowledgments Gaetano Donofrio Dept. of Animal Health Claudio Rivetti Dept. of Biochemistry and Molecular Biology