1. Cationic amphiphilic calixarenes: nanoscopic micelle formation and gene delivery Roman Rodik, Stanislav Miroshnichenko, Vitaly Kalchenko Institute of Organic Chemistry National Academy of Sciences of Ukraine Namrata Jain, Ludovic Richert, Yves Mely, Andrey Klymchenko Laboratory of Biophotonics and Pharmacology, Faculty of Pharmacy, University of Strasbourg

2. Picture from: Calixarene-based multivalent ligands. L. Baldini, A. Casnati, F. Sansone and R. Ungaro Chem. Soc. Rev., 2007, 36, 254–266 Application pathways of calixarenes in biology and medicine

3. Calixresorc[4]arene glycoclusters. DNA binding and transfection Y. Aoyama, Trend. Glycosci. Glycotech. 2005, 17, 39-47.

4. a FuGene ® ; b control; c cone multicalixarene; d 1,3-alt multicalixarene Gen transfection by polycationic multicalixarenes with ammonium residues Results of transfection pDs2-mito (Clontech) plasmid in CHO cells R. Lalor, J. L. DiGesso, A. Mueller and S. E. Matthews, Chem. Commun. 2007, 4907.

5. Gen transfection by tetraguanidinium calixarenes V. Bagnacani, F. Sansone, G. Donofrio, L. Baldini, A. Casnati, R. Ungaro. Org. Lett., 2008, 10, 3953-3956. Results of transfection pEGFP-C1 plasmid in RD-4 cells mediated by calixarene-DOPE formulation

6. Water soluble tetracationic calix[4]arene For tetrapropoxy calix[4]arene CX3 size of aggregates near 3 nm (DLS data) n = 6 N. O. Mchedlov-Petrossyan, L. N. Vilkova, N. A. Vodolazkaya, A. G. Yakubovskaya, R. V. Rodik, V. I. Boyko and V. I. Kalchenko, Sensors 2006, 6, 962. N. O. Mchedlov-Petrossyan, N. A. Vodolazkaya, L. N. Vilkova, O. Y. Soboleva, L. V. Kutuzova, R. V. Rodik, S. I. Miroshnichenko and A. B. Drapaylo, J. Mol. Liq. 2009, 145, 197. CX3

7. Synthesis of tetracationic tetraoctylcalix[4]arenes CX8 CX8im Water solubility of CX8 and CX8im – high!!! 1

8. Pyren fluorescence in CX8 water solution Slow then sharp growth of fluorescence as well as changes in I 1 /I 3 relation indicates that micelles arises in solution I1I1 I3I3

9. CMC values obtained with pyren probe Water 4·10 -4 M5·10 -5 M1·10 -5 M 20 mM Hepes buffer, pH 7.4 7·10 -5 M6·10 -6 M3·10 -6 M Previous and 150 mM NaCl 8·10 -5 M3·10 -6 M2·10 -6 M CX3 CX8CX8im

10. Size of micelles (Data obtained from DLS measurements) Size, nm (polydispersity) Zeta potential, mV 3-4 1 6.3 (0.4)37 6.3 (0.25)41 CX3 CX8 CX8im 1 N. O. Mchedlov-Petrossyan, N. A. Vodolazkaya, L. N. Vilkova, O. Y. Soboleva, L. V. Kutuzova, R. V. Rodik, S. I. Miroshnichenko and A. B. Drapaylo, J. Mol. Liq. 2009, 145, 197. Concentration of CX3 10 -3 M, CX8, CX8im 10 -4 M

11. Fluorescence correlation spectroscopy experiments Sample [NR],  M NSize, nm CX8, 10  M 0.2175.4 0.4295.1 0.850.5 6.6 1.651 6.3 FCS data on CX8 micelles stained with Nile Red

12. Aggregation number of CX8 in micelles FCS Data: 50 particles per excitation volume 0.34×10 -15 L Obtained concentration ca 250 nM. Starting concentration 10 µM. Aggregation number 10 µM/250 nM is 40 Geometrical estimation : CX8 approximately is truncated cone with D 1.53 nm, d 0.51 nm and h 1.62 nm. For micelle formation necessary regular cone geometry with l 2.54 nm. So diameter of micelles 5.1 nm Volume of micelle 69.5 nm 3, volume of CX8 (total cone) 1.5 nm 3, void volume at hexagonal packing near 10%. So the aggregation number (69.5×0.9)/1.5 is 41.7.

13. CT-DNA complexation Ethidium Bromide displacement experiments Tris-bufer 20mM, pH 7.4 [CT-DNA]=210 -5 M Tris-bufer 20mM, pH 7.4, 150mM NaCl [CT-DNA]=210 -5 M Dependence of integral intensity EB at different concentration of cationic calixarenes Normalized graphs

14. SampleParameter CT-DNApDNAZeta potential, mV N/P=2N/P=5N/P=2N/P=5N/P=2N/P=5 CX3 Size (N), nm96212003701300 -15-6 Pld.0.450.570.280.29 CX8 Size (N), nm69586041 42 Pld.0.170.320.140.16 CX8im Size (N), nm72615441 4243 Pld.0.160.240.190.14 CX8/ DOPE Size (N), nm 5750- - Pld. 0.160.18 CX8im/ DOPE Size (N), nm 6650- - Pld. 0.110.17 Size of DNA-calixarene micelles (Data obtained from DLS measurements)

15. AFM image of CT-DNA-calixarene complexes AFM topography (A and C) and phase (B and D) images of calixarene CX8 (A,B) and CX8im (C,D) complexes with CT-DNA (N/P = 2) in 20 mM MES buffer (pH 7). Tapping mode in buffer was used. Circles highlight some larger structures showing, which in phase images can be resolved as combination of smaller particles. V(CX8) 1.5(  0.4)  10 5 nm 3 V(CX8im) 0.9(  0.3)  10 5 nm 3 d(CX8) 65nm d(CX8im) 55 nm

16. DNA-calixarene micelles analysis (CT-DNA was used) One DNA-CX8 micelle contains 700 CX8 micelles

17. Protein biosynthesis Transcription ► Processing and Transport ► Translation Gene transfection Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells. A plasmid (lat. plasmid) is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms

18. Transfection efficiency of calixarene/pDNA complexes in COS-7 cells. Gene expression determined from the luciferase assay was expressed as RLU/mg of total protein. The experiments were repeated three times. Results of pCMV-Luc plasmid transfection mediated by calixarenes and calixarene-DOPE formulations Semi-quantitative analyses, amount of transfected cell in the view area

19. Viability of cells MTT cell viability assay For transfection experiments were used solutions of 10 and 25 µM calixarene concentration

20. Conclusions and perspectives The present results propose a powerful two-step hierarchical approach for generating small DNA nanoparticles. The further development of gene delivery vectors based on calixarenes capable to form stable cationic micellar structures may discover more powerful or selective DNA vectors for needs biology and medicine.

21. Rodik Roman is grateful to ARCUS program (collaboration of Ukraine, Russia and region Alsace, France) for supporting his visit to the French Laboratory. Acknowledgements:

23. Plasmid binding Gel electrophoresis experiments CX3 CX8CX8im

24. Results of GFP plasmid transfection mediated by calixarenes and calixarene-DOPE formulations Semi-quantitative analyses, amount of transfected cell in the view area Transfection efficiency of calixarene/pDNA complexes in COS-7 cells. Gene expression was determined from the count of green fluorescence cells in the quarter of microscope view area.