318
2. La-dipalmitoyl-phosphatidylcholine >99% (TLC) (DPPC—Fig.
1).
3. Deuterium-depleted water (99.99 atom% 16O).
4. 2-HP-β-CD (average Mw ~1460).
3 Methods
1. Obtain
1
H NMR spectra of the samples: (a) Irbesartan dissolved
in D 2 O; (b) 2-HP-β-CD dissolved in D 2 O D 2 O; (c) irbesartan
dissolved in SDS micelles; (d) IRB complexed with 2-HP-β-ΟΗ
and dissolved in D 2 O; (e) IRB complexed with 2-HP-β-ΟΗ and
dissolved in SDS micelles. Pulse sequences are provided in spectrometer libraries.
2. Dissolve 9.8 mg of the complex irbesartan–2-hydroxypropyl-βcyclodextrin in D 2 O in order to obtain NMR spectra. Τhe pulse
sequences included in the libraries of pulse programs were used
in order to acquire 1D
1
H and
13
C NMR spectra.
3. For the preparation of 5 mM of irbesartan in 400 mM SDSd 25 /D 2 O, subject the mixture to sonication in order to provide
a transparent solution.
4. The preparation of the complex irbesartan–2-hydroxypropyl-βcyclodextrin is carried out during a freeze-drying procedure as
it is described in [33]. Specifically, the neutralization method is
applied for the preparation of IRB-2-HP-β-CD aqueous solutions for freeze-drying in a molar ratio (irbesartan/2-HPβ-CD) 1:2. Transfer 408 mg of 2-HP-β-CD and 60 mg of
irbesartan in a 100 μL beaker and suspend them with 50 mL of
water. Subsequently, add small amounts of ammonium hydroxide under stirring, while pH is monitored until complete dissolution and pH adjustment to a value of 9–10. Then, freeze
the resulting solution at −80 °C and freeze-dry using a
Kryodos- 50 model Telstar lyophilizer.
1. Fully hydrate DPPC to form multilamellar bilayers.
2. Record a
13
C cross polarization/magic-angle spinning (
13
C
CP/MAS) solid-state NMR spectra on a 600 MHz spectrometer equipped with a 3.2 mm HX MAS probe. Pack approximately 20 mg of each sample tightly into a zirconia rotor and
spin at 5 kHz. The duration of the CP block in the CP/MAS
experiment should be 5 ms, repetition delay between scans
should be 2 s, and the number of scans 400. The
13
C chemical
shift axis should be referenced to as tetramethylsilane.
For the sample DPPC/HP-β-CD (x = 0.20), record both
13
C CP/MAS and MAS spectra at a sample rotation frequency
of 15 kHz. Record the spectra on a 600 MHz spectrometer
3.1 Investigation
of the Structural
Properties
of Irbesartan
and Irbesartan–2Hydroxypropyl- βCyclodextrin Complex
in Micelles
3.2 The Application
of ssNMR
Spectroscopy to Study
Drug:Membrane
Interactions
Dimitrios Ntountaniotis et al.
2. La-dipalmitoyl-phosphatidylcholine >99% (TLC) (DPPC—Fig.
1).
3. Deuterium-depleted water (99.99 atom% 16O).
4. 2-HP-β-CD (average Mw ~1460).
3 Methods
1. Obtain
1
H NMR spectra of the samples: (a) Irbesartan dissolved
in D 2 O; (b) 2-HP-β-CD dissolved in D 2 O D 2 O; (c) irbesartan
dissolved in SDS micelles; (d) IRB complexed with 2-HP-β-ΟΗ
and dissolved in D 2 O; (e) IRB complexed with 2-HP-β-ΟΗ and
dissolved in SDS micelles. Pulse sequences are provided in spectrometer libraries.
2. Dissolve 9.8 mg of the complex irbesartan–2-hydroxypropyl-βcyclodextrin in D 2 O in order to obtain NMR spectra. Τhe pulse
sequences included in the libraries of pulse programs were used
in order to acquire 1D
1
H and
13
C NMR spectra.
3. For the preparation of 5 mM of irbesartan in 400 mM SDSd 25 /D 2 O, subject the mixture to sonication in order to provide
a transparent solution.
4. The preparation of the complex irbesartan–2-hydroxypropyl-βcyclodextrin is carried out during a freeze-drying procedure as
it is described in [33]. Specifically, the neutralization method is
applied for the preparation of IRB-2-HP-β-CD aqueous solutions for freeze-drying in a molar ratio (irbesartan/2-HPβ-CD) 1:2. Transfer 408 mg of 2-HP-β-CD and 60 mg of
irbesartan in a 100 μL beaker and suspend them with 50 mL of
water. Subsequently, add small amounts of ammonium hydroxide under stirring, while pH is monitored until complete dissolution and pH adjustment to a value of 9–10. Then, freeze
the resulting solution at −80 °C and freeze-dry using a
Kryodos- 50 model Telstar lyophilizer.
1. Fully hydrate DPPC to form multilamellar bilayers.
2. Record a
13
C cross polarization/magic-angle spinning (
13
C
CP/MAS) solid-state NMR spectra on a 600 MHz spectrometer equipped with a 3.2 mm HX MAS probe. Pack approximately 20 mg of each sample tightly into a zirconia rotor and
spin at 5 kHz. The duration of the CP block in the CP/MAS
experiment should be 5 ms, repetition delay between scans
should be 2 s, and the number of scans 400. The
13
C chemical
shift axis should be referenced to as tetramethylsilane.
For the sample DPPC/HP-β-CD (x = 0.20), record both
13
C CP/MAS and MAS spectra at a sample rotation frequency
of 15 kHz. Record the spectra on a 600 MHz spectrometer
3.1 Investigation
of the Structural
Properties
of Irbesartan
and Irbesartan–2Hydroxypropyl- βCyclodextrin Complex
in Micelles
3.2 The Application
of ssNMR
Spectroscopy to Study
Drug:Membrane
Interactions
Dimitrios Ntountaniotis et al.