Ernst Wehrli

Interaction of a lecithin microemulsion gel with human stratum corneum and its effect on transdermal transport

A soybean lecithin microemulsion gel has been studied as a possible matrix for transdermal drug delivery. This gel is transparent and viscous, and it is composed of soybean phosphatidylcholine (lecithin), isopropyl palmitate and a small amount of water. In vitro percutaneous penetration studies of two anti-inflammatory drugs, indomethacin and diclofenac, dissolved in the gel-system resulted in steady state fluxes of about 1 μg h−1 cm−2. In order to estimate the function of the gel as a potential transdermal penetration enhancing system, interaction studies with isolated human stratum corneum were performed using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) as well as low-temperature scanning electron microscopy. These studies indicated that the lecithin gel, in particular isopropyl palmitate, affects the stratum corneum lipid organization even after 1-day incubation (FTIR, DSC), whereas recent in vivo human skin irritation tests showed no significant irritancy.

Direct formation of mixed micelles in the solubilization of phospholipid liposomes by Triton X-100

The vesicle to micelle transition which results in the interaction of the Triton X‐100 surfactant with phosphatidylcholine vesicles was studied by means of dynamic light scattering (at different reading angles) and by freeze‐fracture electron microscopy techniques. Vesicle solubilization was produced by the direct formation of mixed micelles without the formation of complex intermediate aggregates. Thus, vesicle to micelle transformation was mainly governed by the progressive formation of mixed micelles within the bilayer. A subsequent separation of these micelles from the liposome surface (vesicle perforation by the formation of surfactant‐stabilized holes on the vesicle surface) led to a complete solubilization of liposomes.

Preparation and characterization of polyethyl-2-cyanoacrylate nanocapsules containing antiepileptic drugs

Biocompatible and biodegradable colloidal drug delivery systems can be obtained by means of in situ polymerization of alkylcyanoacrylate. In particular, nanocapsules of polyethylcyanoacrylate (PECA) were prepared by adding the monomer to an organic phase, consisting of Miglyol 812 and an organic solvent (ethanol, acetone or acetonitrile), and subsequently mixing the organic phase with an aqueous phase containing Pluronic F68 at different concentrations. The possible mechanism of formation and the influence of preparation conditions on the quality of nanocapsule formulations were investigated by freeze-fracture electron microscopy and laser light scattering using both the inverse Laplace transform and the standard cumulant analysis for data fitting. High-quality nanocapsule systems were obtained using an aprotic fully water-miscible organic solvent such as acetone. The presence of ethanol led to the formation of both nanospheres and nanocapsules. The concentrations of nonionic surfactant in the aqueous phase of monomer in the organic phase did not influence the kind of colloidal suspension obtained. The oil simply plays the role of monomer support. The diameter of PECA nanoparticles (nanospheres and nanocapsules) ranged from 100 to 400 nm. Three antiepileptic drugs (Ethosuximide, 5,5-diphenyl hydantoin and carbamazepine) were entrapped in PECA nanocapsules. The loading capacity of PECA nanocapsules, prepared using acetone as organic solvent, varied from 1% to 11% (drug/dried material) as a function of the solubility (affinity) of the different drugs with the oil core. This parameter also influenced the release from PECA nanocapsules, which was slower for drugs with a higher affinity for Miglyol 812. By encapsulating the three antiepileptic drugs in the PECA nanocapsules, it was possible to achieve controlled drug release. The mechanism of drug release from PECA nanocapsules was mainly diffusion from the oil core through the intact polymer barrier.

A pharmacokinetic and MRI study of unilamellar gadolinium-, manganese-, and iron-DTPA-stearate liposomes as organ-specific contrast agents

Small unilamellar liposomes that contain the lipophilic chelate DTPA-stearate (DTPASA) were used as carriers for the paramagnetic metal ions gadolinium, manganese, and iron. The iron liposomes were unstable in vitro and thus not studied further. The natural targeting properties of these liposomes to the reticuloendothelial system was used in rats and dogs for the imaging of liver and spleen. In vitro incubations with human plasma, followed by high-pressure liquid chromatography (HPLC) separation of the Gd-DTPASA and Mn-DTPASA liposomes showed that after an incubation period of 24 hours, only 4% of the gadolinium was bound to the plasma proteins, whereas, with the Mn-DTPASA liposomes, a transfer of 40% manganese was seen. These results indicate that the Mn-DTPASA complex is not stable. On T1-weighted images, both liposome preparations gave a strong signal enhancement of the organs of the mononuclear phagocyte system (MPS). Gadolinium liposomes accumulated in the liver of rats at a peak concentration 4 hours after application and at a higher concentration compared with the manganese liposomes. Gd-DTPASA liposomes had an elimination half-time from the liver of 61 hours. Manganese liposomes produced stronger contrast at lower concentrations and had faster elimination kinetics from the liver, with a major elimination half-time of 10 hours. Both chelate complexes were eliminated predominantly by the hepatobiliary route. Thus, liposomal Gd-DTPASA appears to be a stable, efficient, and specific magnetic resonance imaging (MRI) contrast agent for the upper abdomen.

Enhanced Therapeutic Effect of Cytidine-5′ -Diphosphate Choline when Associated with GM1 Containing Small Liposomes as Demonstrated in a Rat Ischemia Model

AbstractPurpose. Cytidine-5′-diphosphate choline (CDPc) was encapsulated in long-circulating unilamellar vesicles (SUVs) to improve the drugs biological effectiveness. Methods. SUVs made up of diaplmitoylphosphatidylcholine/diaplmitoylphosphatidylserine/ cholesterol (7:4:7 molar ratio) and 8 mol % of ganglioside GMl were prepared by extrusion through polycarbonate filters (mean diameter 50 nm). The formulation effectiveness was evaluated by an in vivo model of cerebral ischemia on Wistar rats. Results. The enhanced delivery of CDPc into the brain improved the therapeutic effectiveness of the drug. CDPc-loaded SUVs improved the survival rate of ischemized and reperfused Wistar rats (320-350 g) by -66% compared with the free drug. Liposome formulation was also able to effectively protect the brain against peroxidative damage caused by post-ischemic reperfusion. SUVs lowered the conjugated diene levels of the cerebral cortex. The liposomal delivery system did not alter the distribution patterns in the various cerebral lipid fractions of the drug, radiolabeled with 14C-CDPc. Conclusions. CDPc-loaded SUVs were able to protect the brain against damage induced by ischemia. A possible clinical application is envisaged.

Neutrase entrapment in stable multilamellar and large unilamellar vesicles for the acceleration of cheese ripening

We report the encapsulation of neutrase in liposomes for the acceleration of cheese ripening. The liposome preparation procedure consisted of repeated freeze-thaw cycles of multilamellar vesicles followed by extrusion through polycarbonate filters. The neutrase encapsulation efficiency in the liposomes was influenced by the number of freeze-thaw cycles, achieving the highest value after seven cycles. Filtration through 200-nm polycarbonate membranes yielded homogenous size liposome populations with trapping efficiencies of about 65%. The vesicle stability and low neutrase release during the first stages of the cheese-making procedure, coupled with an almost quantitative retention of neutrase-loaded liposomes in cheese curd, ensured a proteolysis rate that was twice that observed in the control cheese.

Three C-phycoerythrin-associated linker polypeptides in the phycobilisome of green-light-grown Calothrix sp. PCC 7601 (cyanobacteria)

Microanalyses by SDS‐PAGE and microsequencing demonstrate that, under green‐light conditions, 3 C‐phycoerythrin associated rod‐linker polypeptides with different N‐terminal amino acid sequences are present in phycobilisomes (PBS) from Calothrix sp. 7601 cells. Two of these polypeptides, corresponding to SDS‐PAGE bands at 36 and 37 kDa, could be assigned, respectively, to the cpeC and epeD genes found on a separate cpeCD‐operon in Calothrix sp. 7601 (Federspiel, N.A. and Grossman, A.R. (1990) J. Bacteriol. 172, 4072‐4081). The third C‐PE rod‐linker polypeptide, LR,2 PE,33, requires, therefore, a third gene with the suggested locus designation ‘cpeE’. A C‐PE (αβ)6‐LR,2 PE,33 complex containing this third rod‐linker polypeptide could be isolated from phycobilisomes and characterized. PBS from both green‐ and red‐light cells of Calothrix contain a single, unique LRC 28 rod‐core linker polypeptide which is not altered during chromatic adaptation.

Thermodynamic stability and osmotic sensitivity of small unilamellar phosphatidylcholine vesicles.

Evidence is presented to show that small unilamellar phosphatidylcholine vesicles with a diameter of approx. 20 nm are osmotically sensitive. Such vesicles respond to osmotic pressure by swelling or shrinking depending on the direction of the applied salt gradient. This is true for small unilamellar vesicles of egg phosphatidylcholine and dimyristoylphosphatidylcholine below and above their crystal-to-liquid crystal transition temperature. At the transition temperature the vesicles are osmotically insensitive due to the increased bilayer permeability resulting in rapid dissipation of salt gradients. Positive salt gradients produce shrinking and collapse of spherical phospholipid vesicles to disks. Shrinking of vesicles is associated with H2O and solute efflux, but only limited solute influx. Clustering of lipid molecules in the bilayers of the resulting disks can be detected by EPR spin labeling. Negative salt gradients produce swelling of vesicles which is associated with H2O and solute influx. Our experiments are consistent with an osmotically perturbed bilayer. In the presence of osmotic gradients the influx and efflux of H2O is coupled with the movement of ions and small molecules which in the absence of salt gradients or osmotic stress cannot pass the phospholipid bilayer. However, during osmotically induced shrinking and swelling of SUV the integrity of the phospholipid bilayer is maintained to the extent that vesicles do not break, and therefore equilibration between external medium and vesicle cavity does not take place.

The effect of monoacylglycerol on the phase behavior of egg phosphatidylcholine

Phosphatidylcholine bilayers can accommodate large quantities of monoacylglycerol. Incorporating up to 40% monoacylglycerol has little effect on the orientation and motion of the phosphatidylcholine polar group. Briefly heating mixed dispersions of 1-monooleoylglycerol/egg phosphatidylcholine (1:1, weight ratio; 2.1:1, mole ratio) to 50-60 degrees C induced spontaneous vesiculation: unilamellar and some oligolamellar vesicles bud off the large multilamellar particles. The size of the resulting vesicles ranges from 100 to 1000 nm, with the bulk of the vesicles having diameters between 100 and 500 nm. The spontaneous vesiculation process is reflected in the visual clearance of the mixed lipid dispersion and in the collapse of the 31P powder NMR spectrum to a sharp, asymmetric peak. The narrowing of the 31P-NMR spectrum is explained in terms of additional molecular and/or segmental motion of the lipid polar groups. In mixed dispersions of 1-monooleoylglycerol/egg phosphatidylcholine containing an excess of 1-monooleoylglycerol (greater than or equal to 50%) domain formation takes place, i.e., the formation of local clusters enriched in either of the two lipids. As a result the mechanical properties of these mixed lipid bilayers seem to be quite different from those of pure egg phosphatidylcholine.

Effect of liposomes on delipidized stratum corneum structure: an ‘in vitro’ study based on high resolution low temperature scanning electron microscopy

Abstract The structural modifications induced in the delipidized stratum corneum (SC) by the addition of SC lipid liposomes were investigated ‘in vitro’ using double-layer for high resolution low-temperature scanning electron microscopy technique (HRLTSEM). Liposomes were prepared from a lipid mixture extracted from the SC with organic solvents and characterized by freeze-fracture electron microscopy. After delipidization and subsequent incubation with liposomes no structural modifications were detected in the SC protein domains. However, the lipid lamellar structure detected in the native SC was lacking after delipidization. The subsequent treatment with liposomes of this delipidized tissue led to the formation of new structures in the intercellular spaces that showed similar organization to the lipid lamellae structure of the native SC. Image analyses of the desmosomes found in the delipidized SC and of the structures formed after treatment with liposomes demonstrated that these new structures did not correspond to desmosomes in spite of the similar appearance of both structures. Hence, the new structures could be associated with a restoration of the damaged lipid lamellae structure of the delipidized SC by the SC lipid liposomes. This effect would involve the interaction between the lipids of the liposomes and the remaining material after delipidization.