Pieter Saveyn

Quantification of hydrophilic ethoxylates in polysorbate surfactants using diffusion H1 NMR spectroscopy.

Polysorbate surfactants (commercially available as Tween) are widely used in pharmaceutical, cosmetic and food products. They are generally considered as esters of ethoxylated sorbitan with fatty acids. Diffusion H1 NMR spectroscopy on a solution of polysorbate 20 in D2O revealed that only one diffusion coefficient was found for the fatty acyl part. Using the Stokes-Einstein equation, it became obvious that this diffusion behavior was caused by micelles. On the other hand, two significantly different diffusion coefficients were found for the methylene groups of ethylene oxide (EO). This indicates the presence of two distinct EO containing species in solution. Since the slowest diffusing EO species has the same diffusion coefficient as the fatty acyl part, it corresponds to the micellar (i.e. fatty acyl bound) ethoxylates. The diffusion coefficient of the fastest diffusing EO species was a factor of four larger than that of the slowly diffusing species and was attributed to water-soluble non-esterified ethoxylates. A solution of polysorbate 20 in the presence of NaOD was prepared to investigate if hydrolysis of the sorbitan ester could be the reason for the occurence of these hydrophilic ethoxylates. It was found that alkaline hydrolysis does lead to an increasing fraction of non-esterified ethoxylates, but is not the cause of its presence in untreated polysorbate samples since these species were also found in solutions of polyethylene glycol oleyl ether (commercially available as Brij), which are not susceptible to hydrolysis. Fractionation of the EO species present in polysorbate 20 into an amphiphilic and a hydrophilic fraction was only partly obtained by activated carbon adsorption. On the other hand, sequential extraction of aqueous polysorbate solutions by ethyl acetate and chloroform enabled a nearly complete fractionation. H1 NMR spectroscopy proved to be very useful since it allows in situ determination of the global composition of a surfactant sample, as well as quantification of both the amphiphilic and hydrophilic ethoxylate fractions via diffusion measurements.

NMR study of the sorption behavior of benzyl alcohol derivatives into sonicated and extruded dioctadecyldimethylammonium chloride (DODAC) dispersions: The relevance of membrane fluidity

The sorption behavior of three benzyl alcohol derivatives with different hydrophobicities into sonicated and extruded DODAC dispersions has been studied using NMR spectroscopy and NMR diffusometry. We show that there is an increased sorption into a sonicated dispersion below the phase-transition temperature (T(m)) as compared to an extruded dispersion. This may be explained by the incomplete lipid chain freezing of charged lipids as a result of the sonication process. Around T(m), a sorption maximum is found that is attributed to the high bilayer disorder under this condition. In addition, a sorption increase and a fluidizing effect at increasing benzyl alcohol derivative concentrations are observed that provide additional evidence for the relevance of the bilayer fluidity on the sorption of hydrophobic components.

Solubilization of flurbiprofen within non-ionic Tween 20 surfactant micelles: a 19F and 1H NMR study.

The solubilization of the poorly water soluble anti-inflammatory drug flurbiprofen in non-ionic Tween 20 surfactant micellar solutions was studied by both (19)F and (1)H NMR spectroscopy in an acidic environment. These non-destructive techniques allowed us to investigate the effect of temperature cycling in situ. Using (19)F NMR, an increased solubilisation capacity was observed as the temperature increased. This effect became more pronounced above the cloud point, which was reduced by more than 30 degrees C in the presence of an excess of flurbiprofen. Upon clouding, peak splitting was observed in the (19)F spectrum, which indicates that two pools of solubilised flurbiprofen exist that are in slow exchange on the NMR frequency timescale. The clouding and solubilization processes were found to be reversible, albeit with slow kinetics. Based on chemical shift differences of both Tween 20 and flurbiprofen, as well as NOESY experiments, the flurbiprofen was found to be accumulated within the palisade layer of the Tween 20 micelles.

Evaluation of the interaction of propranolol with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes: the Langmuir model.

The interaction of the amine containing beta-receptor blocking agent propranolol (Ppn) with dimyristoylphosphatidylcholine (DMPC) vesicles was studied. Using a centrifugation assay, the protonated as well as unprotonated amount of the drug sorbed was verified, whereas the binding of the protonated Ppn was deduced from the surface charge density of the vesicles as calculated from electrophoretic mobility measurements. Assuming a 1:1 binding, a Langmuir model with only two parameters was found to be sufficient to fit all experimental data. Sensitivity analysis revealed that the estimated values of these parameters were reliable and independent from each other. These parameters were truly intrinsic, as electrostatic interactions were accounted for in the model. It was found that the pKa of Ppn shifted from 9.24, when dissolved in water, downward by 1.34 units upon sorption, indicating that the intrinsic partition coefficient of the unprotonated Ppn was about 22 times higher than that of the protonated analog. In addition, a significant increase in the affinity of both Ppn analogs with increasing salt concentration was found. Theoretical analysis revealed that the Langmuir sorption model may be considered as a partitioning model with decreasing partition coefficient as the sorbed amount increases. Thus, the Langmuir model provides a better fit than a simple partition model at conditions that induce a substantial amount of propranolol sorbed, such as high pH and high propranolol concentrations.

Design and Development of Magnetoliposome-Based Theranostics

Magnetoliposomes (MLs) consist of nanometre-sized magnetite cores, enwrapped by a bilayer of phospholipid molecules. In the past we showed that these nanocolloids can be exploited as powerful biocompatible magnetic resonance imaging (MRI) contrast agents. In the present work, we report on the partitioning of the amphiphilic drug, (R/S)-propranolol, within the lipidic envelope of MLs, built up of the zwitterionic dimyristoylphosphatidylcholine. Furthermore, it is also shown that MLs are easily internalized by 3T3 fibroblasts, used in this study as a representative cell model, without damaging cell viability. Overall, the results deliver the proof-of-concept that drug-loaded MLs have great potential as unique intracellular theranostics, i.e., as a nanoscale delivery system with combinatory therapeutic-diagnostic imaging modalities.

Sedimentation and resuspendability evaluation of pharmaceutical suspensions by low-field one dimensional pulsed field gradient NMR profilometry

In order to study the sedimentation of pharmaceutical suspensions using low-field one dimensional pulsed field gradient nuclear magnetic resonance (1D pfg NMR) profilometry, the accuracy of signal acquisition as well as the spatial resolution of a commercial spectrometer operating at 23.4 MHz was investigated. The use of a solid Teflon spacer revealed that the accuracy of signal acquisition was independent of spatial position (height). The standard deviation of distance determinations was less than 150 µm, whereas the accuracy of water content determination was within 2% in the central part of the detection zone and deteriorated to 4% in the outer parts. The study of aqueous paramagnetic MnCl2 solutions indicated an exponential relationship between the relative signal intensity and the transverse relaxation decay constant. From this relationship, the relative water content of suspensions could be derived from their signal intensity relative to that of water. Using concentrated paliperidone palmitate dispersions as model suspensions, low-field 1D pfg NMR profilometry has been proven to be suitable for the evaluation of both the sedimentation and resuspendability behavior of viscous, opaque suspensions, for which visual detection of homogeneity may be difficult.

Enclosed volume determination of concentrated dioctadecyldimethylammonium chloride (DODAC) vesicular dispersions by low-resolution proton NMR diffusometry and T-2 relaxometry

The enclosed volume of concentrated dioctadecyldimethylammonium chloride (DODAC) dispersions has been determined by means of low-resolution NMR pfg-diffusometry and T(2) relaxometry. The pfg-NMR diffusometry method is based on the different diffusion behaviors of water in the external and internal phases and as such does not require the addition of a tracer. On the other hand, T(2) relaxometry is based on the different relaxation behaviors of water fractions upon addition of manganese chloride as external (paramagnetic) probe. It was noticed that reliable results are found only for temperatures below the phase transition temperature of DODAC, when the exchange between the two water compartments can be neglected. At 5 °C, these two independent methods resulted in similar enclosed volume values, meaning that the results are reliable and reflect the real enclosed volume. In addition, the T(2) relaxometry method has been proven to be useful in the investigation of the DODAC membrane permeability.

Influence of the vesicular bilayer structure on the sorption of ethylbenzyl alcohol

The influence of the physicochemical properties of the vesicular bilayer on the sorption of poorly water soluble compounds was investigated with pulsed field gradient 1H nuclear magnetic resonance (PFG-NMR) for the case of phosphatidylcholine and dioctadecyldimethylammonium bromide (DODAB), using 4-ethylbenzyl alcohol as a model compound. Hereby, the effect of bilayer thickness at a constant physicochemical state was studied using a range of phosphatidylcholines of varying chain lengths, whereas DODAB was preferred to check the influence of the bilayer physicochemical state since this cationic lipid is characterized by three different states within the studied temperature range. When the phospholipid alkyl chain length was changed, no differences were observed in the sorption which was linked to the surface-mediated sorption. On the other hand, when the chemical composition was preserved but the temperature and thus the physical state of the bilayer were changed, the sorption in dioctadecyldimethylammonium bromide (DODAB) vesicles changed dramatically. From those experiments, a strong relationship between the ordering of the surfactant molecules and the sorption can be assumed.