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CALIXARENE-BASED NANOSYSTEMS FOR GENE DELIVERY

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. Vettore. Vettore. Complesso. Complesso.

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CALIXARENE-BASED NANOSYSTEMS FOR GENE DELIVERY

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  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. Vettore Vettore Complesso Complesso Vettore Vettore - - DNA DNA DNA DNA terapeutico terapeutico Membrana cellulare Membrana cellulare Endosoma Endosoma Sintesi delle Sintesi delle proteine proteine Nucleo Nucleo “Gene Therapy” Delivery of nucleic acids to patients for therapeutic purposes Vector Vector Vector-DNA complex Vector-DNA complex Therapeutic DNA 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) Endosome Protein Synthesis Nucleus

  3. 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). VIRAL VECTORS Possible infection risk (remove as many genes as possible) Immune reaction (memory from previous infections) Protect therapeutic genes well Show some specificity SYNTHETIC NON-VIRAL VECTORS No hazard from native gene expression Less efficient gene transfer than viruses Lower tissue specificity Liposomes Cationic Polymers Gemini Surfactants Glycocluster Nanoparticles Adenovirus / Adeno-associated Virus Retroviral Vectors Herpes Simplex Virus Lentiviral Vectors

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

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

  6. Synthesis

  7. Water solubility  10-4

  8. GenePORTER GenePORTER transfection transfection 4G4Oct 4G4Oct - - 4G4Oct 4G4Oct - - 4G4Oct 4G4Oct - - 4G4Oct 4G4Oct - - cone cone cone cone cone cone cone cone reagent reagent a a m m 5 5 M M m m m m m m 10 10 M M 20 20 M M 40 40 M M 4G4Pr 4G4Pr 4G4Pr - - - 4G4Pr 4G4Pr 4G4Pr - - - 4G4Pr 4G4Pr 4G4Pr - - - 4G4Pr 4G4Pr 4G4Pr - - - DOPE DOPE DOPE cone cone cone cone cone cone cone cone cone cone cone cone b b m m m m m m m m m m 15 15 M M 15 15 M M 15 15 M M 15 15 M M 30 30 M M + + + + + + m m m m m m DOPE 15 DOPE 15 M M DOPE 30 DOPE 30 M M DOPE 7.5 DOPE 7.5 M M Cell Transfection

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

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

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

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

  13. Transfection ! =

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

  15. Transfection ! + DOPE =

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

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

  20. 1H NMR spectra in D2O (300 MHz, 300 K)

  21. 10.0 mM 5.0 mM 2.5 mM 1.6 mM cmc 1.0 mM 0.5 mM 0.2 mM 0.2 mM Dilution experiments by 1H NMR in D2O (300 MHz, 300 K) with 4G4Hex-cone

  22. LogD LogD LogD NMR DOSY experiments with 4G4Hex-conein D2O (300 MHz, 300 K) Hydrodynamic radius rH of monomer: 8.9 Å Hydrodynamic radius rH of aggregate: 32.7 Å

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

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