The final lipid concentration was 50mM, while CYSP/DMPC in mixed systems was 6% M/M as described in previous studies. Various W/W proportions of
DMPC to POLYA (from 3 to 12) and POLYA-CYSP complexes (from 3 to 15) were tested. The results presented here used 4/50 complexes to DMPC and 3/50 POLYA to DMPC weight ratios. The same procedures were used to prepare multilayers for 2H-NMR experiments, except that 25% DMPC with perdeuterated chains was used (DMPC-d54) to prepare the liposomes. 2.3. Methods 2.3.1. NMR Experiments 1H-NMR experiments were recorded Inhibitors,research,lifescience,medical at 295K on a Brüker AVANCE III-400 spectrometer using a presaturation of the water resonance and a spectral width of 10ppm. As preliminary relaxation studies evoked T1 values around 0.6s, a recycling delay of 2.5s between pulses was used with π/3 pulses (4.8μs). The chemical shifts were referenced by setting the water resonance at 4.75ppm. 1H-NMR attribution was considered in reference to natural alpha-cyclodextrin and controlled Inhibitors,research,lifescience,medical by Inhibitors,research,lifescience,medical standard correlation spectroscopy experiments
[13]. The first recordings of the POLYA/CYSP complex showed chemical shift variations with respect to POLYA, suggesting that a molecular association operating under fast Gemcitabine in vitro exchange kinetics conditions was present. Using its very coarse approximation of a complex formation, the classical method described by Job [16–18] was used to extract an apparent macroscopic stoichiometry of the complex, while the SIMPLEX mathematic determination method (EXPREX or MURIEL-X algorithms generously provided by Bruno Perly, CEA Saclay, France) gave estimations of the apparent Inhibitors,research,lifescience,medical association constant [19]. 31P-NMR experiments were performed at 162MHz. Phosphorus spectra were recorded using a dipolar echo sequence (π/2-t-π-t) with a t value of 12μsec, a recycling delay of 2.5s, and a composite proton
decoupling. Phosphoric acid (85%) was used as external reference. 2H-NMR experiments Inhibitors,research,lifescience,medical were performed at 61MHz. Deuterium spectra were recorded using a quadrupolar echo sequence (π/2-t-π/2-t) with a t value of 15μsec and a 10s recycling delay. The free induction decay was shifted in fractions of the dwelling time to ensure that the effective time for the Fourier transform corresponded to the top of the echo [20]. The sample temperature was regulated within 1°C by a BVT-1000 unit. 2H-NMR spectra treatment: DNA ligase in order to extract suitable quadrupolar splitting measurements (ΔνQ), the spectra were de-Paked according to the Seelig procedure [21]. This allowed a fluidity profile to be built and calculation of the carbon-deuterium bond segmental order parameter SCD using the following classical relation [11, 20]: SCD=3cos2β−12, (1) where β is the average angle between the carbon deuterium bond and the direction perpendicular to the bilayer normal.