1.5 Aim of the
Chapter
The aim of this chapter is to describe methods for the efficient
delivery of miRNA-targeting PNAs using that amphiphilic tetraargininocalix[4]arene as vector.
Considering that this vector is not commercially available, in
the Methods section the synthetic procedures for the calixarene
derivative are reported together with those relative to the production of the complexes between vector and PNA, to the transfection,
the uptake analysis, the evaluation of the transfection effects and the
cell viability studies study.
As a model experimental system PNA-based miR-221-3p targeting was chosen [34, 46, 48] using human glioma U251
cells [64].
2 Materials
2.1 Argininocalix[4]
arene 1
For the synthesis of the vector, the commercially available materials
used in the Methods section are:
1. p-Tert-butylcalix[4]arene.
2. N,N
0
-Dimethylformamide (DMF).
3. Sodium hydride (NaH, 60% in oil).
4. 1-Iodohexane.
5. Chloridric acid (HCl).
6. Dichloromethane (DCM).
7. Methanol (MeOH).
8. Sodium nitrate (NaNO 3 ).
9. Trifluoroacetic acid (TFA).
10. Magnesium sulfate (MgSO 4 ).
11. Hydrazine hydrate (NH 2 NH 2 ·H 2 O).
12. Pd 10% on charcoal (Pd/C).
13. N α -Boc-N ω -Pbf-L-Arg.
14. N,N
0
-dimethylaminopyridine (DMAP).
15. Hydroxybenzotriazole (HOBt).
16. N-ethyl-N
0 -(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC).
17. Sodium hydrogen carbonate (NaHCO 3 ).
18. Triisopropylsilane (TIS).
19. Ethyl acetate (AcOEt).
20. Thin-layer chromatography (TLC) plates: 60 F254 silica gel
and 60 RP-18 silica gel.
21. 230–400 mesh silica gel for column chromatography.
126
Alessia Finotti et al.
Chapter
The aim of this chapter is to describe methods for the efficient
delivery of miRNA-targeting PNAs using that amphiphilic tetraargininocalix[4]arene as vector.
Considering that this vector is not commercially available, in
the Methods section the synthetic procedures for the calixarene
derivative are reported together with those relative to the production of the complexes between vector and PNA, to the transfection,
the uptake analysis, the evaluation of the transfection effects and the
cell viability studies study.
As a model experimental system PNA-based miR-221-3p targeting was chosen [34, 46, 48] using human glioma U251
cells [64].
2 Materials
2.1 Argininocalix[4]
arene 1
For the synthesis of the vector, the commercially available materials
used in the Methods section are:
1. p-Tert-butylcalix[4]arene.
2. N,N
0
-Dimethylformamide (DMF).
3. Sodium hydride (NaH, 60% in oil).
4. 1-Iodohexane.
5. Chloridric acid (HCl).
6. Dichloromethane (DCM).
7. Methanol (MeOH).
8. Sodium nitrate (NaNO 3 ).
9. Trifluoroacetic acid (TFA).
10. Magnesium sulfate (MgSO 4 ).
11. Hydrazine hydrate (NH 2 NH 2 ·H 2 O).
12. Pd 10% on charcoal (Pd/C).
13. N α -Boc-N ω -Pbf-L-Arg.
14. N,N
0
-dimethylaminopyridine (DMAP).
15. Hydroxybenzotriazole (HOBt).
16. N-ethyl-N
0 -(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC).
17. Sodium hydrogen carbonate (NaHCO 3 ).
18. Triisopropylsilane (TIS).
19. Ethyl acetate (AcOEt).
20. Thin-layer chromatography (TLC) plates: 60 F254 silica gel
and 60 RP-18 silica gel.
21. 230–400 mesh silica gel for column chromatography.
126
Alessia Finotti et al.