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«AMINO- AND GUANIDINOGLYCOSIDE-BASED VECTORS FOR GENE AND DRUG DELIVERY Doctoral Dissertation of: Aurora Sganappa I.D. 802585 Supervisor: Prof. ...»

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POLITECNICO DI MILANO

DEPARTMENT OF CHEMISTRY, MATERIALS AND CHEMICAL

ENGINEERING “GIULIO NATTA”

DOCTORAL PROGRAMME IN

INDUSTRIAL CHEMISTRY AND CHEMICAL ENGINEERING

AMINO- AND GUANIDINOGLYCOSIDE-BASED

VECTORS

FOR GENE AND DRUG DELIVERY

Doctoral Dissertation of:

Aurora Sganappa I.D. 802585

Supervisor:

Prof. Alessandro Volonterio

The Chair of the Doctoral Program:

Prof. Frassoldati Alessio 2012-2015 XXVIII Cycle “Every time you decide, there is a loss, no matter how you decide. It is always a question of what you cannot afford to lose.” Francisco X. Stork II Acknowledgements First and foremost, I would like to express my special appreciation and thanks to my advisor Professor Alessandro Volonterio, for being my mentor in these three years, for encouraging my research and for allowing me to grow as a research scientist. I appreciate all his contributions of time, ideas, teachings and advice to make my Ph.D. experience productive and stimulating. His patience, continuous support and guidance helped me in all the time of research and writing of this Ph.D. thesis.

I would also like to thank all the members of Prof. Volonterio’s research group: Dr.

Alessandra Ghilardi, Dr. Monica Sani, Dr. Fiorenza Viani, Dr. Massimo Frigerio, Dr. Maria Cristina Bellucci, Dr. Bianca Rossi and Dr. Chiara Pennetta who have contributed immensely to my personal and professional time at Polytechnic of Milan. All of them have been a source of good advice and collaboration, helpful and friendly with me, and I enjoyed all the coffee breaks and lunches spent together.

Besides my advisor and his research group, I would like to thank Prof. Ling Peng, Prof. Francesco Sansone and Prof. Yitzhak Tor for their interesting collaborative projects, which provided me the core of this experimental work, and for their insightful comments and suggestions. Moreover, I would to thank them for being part of my committee and for letting my defense be an enjoyable moment.

I am very grateful to Prof. Tor for give me the opportunity to improve my expertise and competencies and to work with his research group at UCSD. I appreciate their advice about my research as well as their willingness and time spent to show me how some type of biological tests work.

I would like to acknowledge the Prof. Gabriele Candiani and his team for the important and fruitful collaboration and for their helpfulness and time.

–  –  –

I would like to thank all the persons, friends, acquaintances, coworkers and so on, that helped me in any way; I appreciate. I would also acknowledge all the situations and persons, which I ran on my road, I learned something different or new from everthinhg and everyone, and this make me, along with my choices, my decisions and my experiences, the person I am today.

A special thanks to Salvatore who with patience, suffered me and supported me during this writing period. Thanks for everything you have done for me.

At the end, I would like to thank you that you are spending your time reading this thesis, this makes me proud and I hope you find it useful.

–  –  –

Bellucci M.C., Sani M., Sganappa A., Volonterio A., “Diversity Oriented Combinatorial Synthesis of Multivalent Glycomimetics Through a Multicomponent Domino Process”, ACS Comb. Science 2014, 16, 711-720.

Bellucci M.C., Sganappa A., Volonterio A., “Multicomponent Diversity Oriented Synthesis of Multivalent Glycomimetics Containing Hexafluorovaline”, Tetrahedron, 2015, 71, 7630-7637.

Sganappa A., Volonterio A., Wexselblatt E., Tor Y., “Biotin-PAMAM-GuanidinoNeomycin Conjugates: Synthesis and Cellular Uptake” 2015, ISBN 978-88-86208-97-0.

Sganappa A., Volonterio A., Tor Y., “Synthesis of Novel Derivatives of Aminoglycosides as Antibiotics by a Domino Multi Component Process” 2015, ISBN 978-88-86208-97-0.

–  –  –

List of Figures List of Schemes List of Tables List of Abbrevations Abstract Chapter 1: Introduction

1.1 Gene Delivery

1.1.1 Viral-based Delivery Systems

1.1.2 Non–viral based Delivery Systems

1.2 Cationic Polymers and Dendrimers

1.2.1 Polyethylenimine (PEI)

1.2.2 Polyamidoamine (PAMAM)

1.3 Drug Delivery

1.3.1 Drug Carriers

1.4 Aminoglicosides

1.4.1 Chemical Structure

1.4.2 Antibacterial activity

1.4.3 Structure-activity relationship (SAR)

1.4.4 Guanidinoglycosides

Chapter 2: Background and Aims

2.1 Gene Delivery Project

–  –  –

2.1.2 Background and Prospect of the Project

2.2 Drug Delivery Project

2.2.1 Aim of the Project

2.2.2 Background and Prospect of the Project

2.3 Novel Antibiotics Project

2.3.1 Aim of the Project

2.3.2 Background and Prospect of the Project





Chapter 3: Results and Discussions

3.1 Gene Delivery Project

3.1.1 PAMAM G4-Aminoglycoside Conjugates:

Synthesis and Biological Activity

3.1.2 PAMAMs-Neomycin and GuanidinoNeomycin Conjugates: Synthesis and Biological Activity………………………………………………………..

3.1.3 Synthesis of calix[4]arene-aminoglicoside Conjugates

3.1.4 Hybrid lipodendrimers-aminoglicoside Conjugates:

3.2 Drug Delivery Project

3.2.1 Biotin-PAMAMs-Guanidinoglycoside Conjugates:

3.3 Novel Antibiotics Project

3.3.1 Sugar-Neomycin Conjugates:

Chapter 4: Conclusions

4.1 Gene Delivery Project

4.2 Drug Delivery Project

4.3 Novel Antibiotics Project

Chapter 5: Experimental Section

5.1 Gene Delivery Project

–  –  –

5.1.2 PAMAMs-Amino and Guanidinoglycosides Conjugates:

5.1.3 Synthesis of Calix[4]arene-aminoglycoside Conjugates

5.1.4 Hybrid lipo-dendrimers-aminoglycoside Conjugates

5.2 Drug Delivery Project

5.3 Novel Antibiotics Project

Chapter 6: References

–  –  –

Figure 1. Overview of Gene Therapy

Figure 2. Schematic generalized representation of delivery of a DNA-based therapeutic using a viral or non-viral DNA delivery vector: (1) complexation and/or entrapment; (2) interaction with cell membrane; (3) cellular internalization mediated by endocytosis; (4) endosomal breakdown; (5) cytoplasmic release of DNA-based therapeutic-vector complex or DNA-based therapeutic; (6) dissociation of DNA from vector; (7) nuclear translocation.

Figure 3. Scheme of convergent synthesis of PAMAM dendrimers with EDA and methyl acrylate.

Figure 4. Structures of PAMAMG2 and PAMAMG4 dendrimers.

Figure 5. Examples of functionalized PAMAM.

Figure 6. Structures of Aminoglicosides.

Figure 7. Structures of three different Neomycins.

Figure 8. Schematic structure of Aminoglycosides 4,5-disobstituted and 4,6-disubstituted.

The aminocyclitol ring is evidenced in blue.

Figure 9. Structures of clinically useful atypical aminoglycosides.

Figure 10. Structures of 1,3 diaminoinositol groups, the pharmacophores of aminoglycoside antibiotics.

Figure 11. A.

Three-dimensional structure of the paromomycin (3) complex with an oligonucleotide, represented by the bacterial decoding-site internal of helix 44 in 16S

rRNA. Paromomycin is represented in ball and stick (light blue: carbon, dark blue:

nitrogen and red: oxygen) and rRNA in stick (green: carbon, dark blue: nitrogen and red:

oxygen). B. Main interactions between paromomycin and the target sub-domain of 16S rRNA.[84]

Figure 12. (a) Secondary structure of the aminoglycoside-binding pocket in helix 44 of 16S rRNA.

Key polymorphic residues determining the selectivity of aminoglycosides are residues 1408 and 1491, highlighted in bold green. (b) Overview of paromomycin bound IX to the bacterial A site and detailed view of the 6’ OH paromomycin ring-I stacking interaction with G1491 and hydrogen bonding with A1408 and A1493. Hydrogen bonds between aminoglycoside ring I and A1408 are shown as red dotted lines, as is hydrogen bonding between 4’OH and O2P of A1493.[87]

Figure 13. Structures of two amino-aminoglycoside and their natural anologs.

............... 44 Figure 14. Structure of PAMAMG4-Neamine, PAMAMG4-Paramomycin, PAMAMG4Neomycin Conjugates.

Figure 15. Figure 1.

1H NMR spectra recorded in D2O of PAMAM G4 dendrimer (red spectrum), paramomycin (green spectrum), and PAMAM G4−paromomycin conjugate (blue spectrum).

Figure 16. 1H NMR spectra recorded in D2O of PAMAM G4 dendrimer (red spectrum), neomycin (green spectrum), and PAMAM G4−neomycin conjugate (blue spectrum).

...... 59 Figure 17. 1H NMR spectra recorded in D2O of PAMAM G4 dendrimer (red spectrum), neamine (green spectrum), and PAMAM G4−neamine conjugate (blue spectrum).......... 59 Figure 18. The MALDI spectra of: A) PAMAM G4 dendrimer, peak at 13226 Da corresponds to the single charged ion of the dendrimer; B) PAMAM G4-paromomycin 30, peak at 24600 Da corresponds to the double charged ion of the PAMAM conjugate; C) PAMAM G4-neomycin 31, peak at 24580 Da corresponds to the double charged ion of the PAMAM conjugate

Figure 19. DNA complexation ability of PAMAMG4-Aminoglycosides Conjugates 29-31.

. 61 Figure 20. Transfection efficiency and cytotoxicity of PAMAM G4 and PAMAM G4derivatives in HeLa cells. (A) Transfection efficiency and (B) cytotoxicity of polyplexes prepared with pGL3 at different N/P were evaluated after incubation for 48 h in 10% FBS.

(C) Influence of FBS content on the transfection efficiency of polyplexes prepared at N/P 15. (D) Comparative cytotoxicity assay ofPAMAMG4 dendrimer andPAMAMG4derivatives 29−31 delivered as polyplexes at N/P 15 and free in solution at equivalent concentration. 25 kDa bPEI was utilized at N/P 10.

Figure 21. Results of t ransfection efficiency and cytotoxicity of PAMAM G4 and PAMAM G4-derivatives in U87-MG cells.

(A) Transfection efficiency and (B) cytotoxicity of polyplexes prepared with pGL3 at different N/P were evaluated after incubation for 48 h X in 10% FBS. (C) Influence of FBS content on the transfection efficiency of polyplexes prepared at N/P 15. (D) Comparative cytotoxicity assay ofPAMAMG4 dendrimer andPAMAMG4-derivatives 29−31 delivered as polyplexes at N/P 15 and free in solution at equivalent concentration. 25 kDa bPEI was utilized at N/P 10.

Figure 22. Results of transfection efficiency and cytotoxicity of PAMAM G4 and PAMAM G4-derivatives in Cos-7 cells.

(A) Transfection efficiency and (B) cytotoxicity of polyplexes prepared with pGL3 at different N/P were evaluated after incubation for 48 h in 10% FBS.

(C) Influence of FBS content on the transfection efficiency of polyplexes prepared at N/P 15. (D) Comparative cytotoxicity assay ofPAMAMG4 dendrimer andPAMAMG4derivatives 29−31 delivered as polyplexes at N/P 15 and free in solution at equivalent concentration. 25 kDa bPEI was utilized at N/P 10.

Figure 23. Results of Antibacterial activity of PAMAMG4-Neamine, PAMAMG4Paramomycin, PAMAMG4-Neomycin Conjugates 29-31 in E.

Coli (A,C,E) and S. Aureus (B,D,F) bacteria.

Figure 24. Bacterial killing assay of dPAMAM G4-derivatives 30 and 31, paromomycin, neomycin, and unmodified dPAMAM G4

Figure 25. Antibacterial activity of polyplexes and free polymers

Figure 26. Tranfection efficiency, cytotoxicity and antibacterial activity of PAMAMG4, PAMAMG4-neomycin 31 and PAMAMG4-paromomycin 30 poliplexes.

Figure 27. Structure of PAMAMG4-Neomycin, PAMAMG2-Neomycin, PAMAMG4GuanidinoNeomycin and PAMAMG7-Neomycin conjugates.

Figure 28. 1H NMR spectra recorded in D2O at 400 Mhz and 305 K of a) PAMAMG2neomycin conjugate 69; b) PAMAMG4−neomycin conjugate 65; c) PAMAMG4−guanidinoneomycin conjugate 68 and d) ) PAMAMG7−neomycin conjugate 70.

Figure 29. The MALDI spectra of: a) PAMAMG2-neomycin conjugate 69, peak at 16030 Da corresponds to the single charged ion; b) PAMAMG4-neomycin conjugate 65, peak at 39493 Da corresponds to the repeated 40 neomycin-linker grafted; c) PAMAMG4guanidinoneomycin conjugate 68, peak at 31776 Da corresponds to PAMAMG4 plus 15 guanidinoneomycin-linker tethered

XI Figure 30. DNA complexation of all free PAMAMs generatios, PAMAMG2-Neomycin, PAMAMG4-Neomycin, PAMAMG7-Neomycin and PAMAMG4-Guanidinoneomycin Conjugates.

Figure 31. Preliminary results of Transfection efficiency of PAMAMG2-Neomycin, PAMAMG4-Neomycin, PAMAMG7-Neomycin and PAMAMG4-Guanidinoneomycin Conjugates in HeLa cells.

Figure 32. Preliminary results of Cytotoxicity of PAMAMG2-Neomycin, PAMAMG4Neomycin, PAMAMG7-Neomycin and PAMAMG4-Guanidinoneomycin Conjugates in HeLa cells.

Figure 33. Transfection efficiency of PAMAM G2,G4,G7, PAMAMG2-Neomycin, PAMAMG4-Neomycin, PAMAMG7-Neomycin and PAMAMG4-Guanidinoneomycin Conjugates in COS-7cells.

Figure 34. Cytotoxcicity of PAMAM G2,G4,G7, PAMAMG2-Neomycin, PAMAMG4Neomycin, PAMAMG7-Neomycin and PAMAMG4-Guanidinoneomycin Conjugates in COScells.

Figure 35. Kanamycin-cholesterol (kanaChol-6’) and triguanidinokanamycin-carbomylcholesterol (TGKC).

Figure 36. Cholesterol-neomycin (CholNeo) and cholesterol-(paromomycin Chol-Paromo).



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