2.8 Apoptosis Assay
1. Annexin V and Dead Cell assay (Luminex Corporation) to
evaluate possible pro-apoptotic effects of PNA.
2. Muse Cell Analyzer instrument (Millipore Corporation) to
perform Annexin V assays.
2.9 Statistics
Results are expressed as mean Æ standard error of the mean (SEM).
Comparisons between groups were made by using paired Student’s
t test and a one-way analysis of variance (ANOVA). Statistical
significance was defined with p < 0.01.
3 Methods
3.1 Preparation of
the Argininocalix[4]
arene 1
All the following reactions (Fig. 2) are performed in N 2 atmosphere. All the procedures are performed under a fume hood.
3.1.1 Synthesis
butyl-25,26,27,28tetrahexyloxycalix[4]
arene (2)
1. Into a two-necked round-bottom flask suspend p-tert-butylcalix[4]arene (2.5 g, 3.85 mmol) in dry DMF (40 mL), stir and
cool at 0
C with an ice bath.
2. Add NaH (60% in oil, 1.85 g, 12 equivalent) to the stirring
mixture (CAUTION! Hydrogen develops at this stage).
3. After 30 min add 1-iodohexane (6.70 mL, 12 equivalent) to
the reaction and, after additional 30 min, remove the ice bath
leaving the reaction to proceed at room temperature for 24 h
(see Note 2).
Fig. 2 Synthetic pathway for the preparation of argininocalix[4]arene 1
PNA Delivery with Argininocalix[4]arene
129
1. Annexin V and Dead Cell assay (Luminex Corporation) to
evaluate possible pro-apoptotic effects of PNA.
2. Muse Cell Analyzer instrument (Millipore Corporation) to
perform Annexin V assays.
2.9 Statistics
Results are expressed as mean Æ standard error of the mean (SEM).
Comparisons between groups were made by using paired Student’s
t test and a one-way analysis of variance (ANOVA). Statistical
significance was defined with p < 0.01.
3 Methods
3.1 Preparation of
the Argininocalix[4]
arene 1
All the following reactions (Fig. 2) are performed in N 2 atmosphere. All the procedures are performed under a fume hood.
3.1.1 Synthesis
butyl-25,26,27,28tetrahexyloxycalix[4]
arene (2)
1. Into a two-necked round-bottom flask suspend p-tert-butylcalix[4]arene (2.5 g, 3.85 mmol) in dry DMF (40 mL), stir and
cool at 0
C with an ice bath.
2. Add NaH (60% in oil, 1.85 g, 12 equivalent) to the stirring
mixture (CAUTION! Hydrogen develops at this stage).
3. After 30 min add 1-iodohexane (6.70 mL, 12 equivalent) to
the reaction and, after additional 30 min, remove the ice bath
leaving the reaction to proceed at room temperature for 24 h
(see Note 2).
Fig. 2 Synthetic pathway for the preparation of argininocalix[4]arene 1
PNA Delivery with Argininocalix[4]arene
129