Mitogenic activity of high molecular polysaccharide fractions isolated from the Cupressaceae Thuja occidentale L. I. Macrophage-dependent induction of CD-4-positive T-helper (Th+) lymphocytes.

The arborvitae or Thuja occidentale L., one of the Cupressaceae, has rarely been investigated until now. Several authors have demonstrated that allopathic extracts of this plant could be used as strong antiviral agents directed against plant and animal viruses. Polysaccharide fractions with molecular weights ranging between 20,000 and 1,000,000 and higher have been isolated from the aqueous alkaline extract of the herbal parts of T. occidentale L. by ethanol precipitation and fractionation by ultrafiltration. High molecular subfractions of Thujapolysaccharides (TPS) proved to be highly mitogenic on peripheral blood leukocytes. It was demonstrated by the alcalic immune phosphatase-antiphosphatase and Pappenheim staining methods that the mitogenic and cluster-forming activity of TPS cause T cell induction, in particular, of CD 4-positive T-helper/inducer cells as opposed to B cells. The CD-4+ T-helper/inducer cell induction is connected to an increased production of IL-2. The cluster-forming ability and mitogenity of TPS correlates well with the 3H-thymidine uptake and seems to be IL-1 and IFN-gamma dependent as could be shown by blocking the mitogenic effect using anti-IL-1- and anti-IFN antibodies. Whether it is possible to use these polysaccharide fractions as an adjuvant in the therapy of immune deficiency syndromes and cancer must now be further investigated.

Mitogenic activity of high molecular polysaccharide fractions isolated from the cuppressaceae Thuja occidentalis L. enhanced cytokine-production by thyapolysaccharide, g-fraction (TPSg).

Thuja polysaccharide g fraction (TPSg) was shown to be an inducer of the CD4+ fraction of the human peripheral blood T-cell subset (1,2). Furthermore, it could be demonstrated that TPSg is a potent inhibitor of the expression of HIV-1-specific antigens and of the HIV-1-specific reverse transcriptase (3). This report deals with the cytokine pattern induced by TPSg in human peripheral blood lymphocyte (PBL) and purified monocyte/macrophage cultures. In addition, a further characterization of the CD4+ T-cell fraction stimulated by TPSg was performed by FACS analysis. TPSg is induces IL-1 beta, IL-2, IL-3, IL-6, gamma-IFN, G-CSF, GM-CSF, and TNF-beta production in PBL cultures; and IL-1 beta and IL-6 in monocyte/macrophage cultures. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that no IL-4 was produced by PBL cultures under TPSg influence.

Rheology of nanostructured lipid carriers (NLC) suspended in a viscoelastic medium.

Colloidal lipid nanoparticle dispersions have been characterized by rheological measurements using two different nanostructured lipid carrier (NLC)-based formulations intended for cosmetic application of substances like sunflower oil and alpha-tocopherol. This study has shown that rheological and viscoelastic properties of aqueous NLC dispersions are quantitatively very different depending on the composition of the oil phase and the temperature of storage despite similar or even identical particle size. NLC were loaded with 30% active ingredient relative to the particle mass. Stearyl alcohol was used as lipid matrix and the particle sizes determined by photon correlation spectroscopy were in the range 210-270 nm. In general, sun flower oil-loaded NLC dispersions showed distinctly higher storage modulus (G'), loss modulus (G") and complex viscosity (eta*). Storage at lower temperature (4 degrees C versus 20 degrees C) delay the build up of a microstructure affected not only by size and stabilizer but also loaded ingredient and storage history after preparation, i.e. storage at room temperature accelerates the build up of a final suspension structure.

Vitamin A loaded solid lipid nanoparticles for topical use: occlusive properties and drug targeting to the upper skin.

To evaluate the potential use of solid lipid nanoparticles (SLN) in dermatology and cosmetics, glyceryl behenate SLN loaded with vitamin A (retinol and retinyl palmitate) and incorporated in a hydrogel and o/w-cream were tested with respect to their influence on drug penetration into porcine skin. Conventional formulations served for comparison. Excised full thickness skin was mounted in Franz diffusion cells and the formulations were applied for 6 and 24 h, respectively. Vitamin A concentrations in the skin tissue suggested a certain drug localizing effect. High retinol concentrations were found in the upper skin layers following SLN preparations, whereas the deeper regions showed only very low vitamin A levels. Because of a polymorphic transition of the lipid carrier with subsequent drug expulsion following the application to the skin, the drug localizing action appears to be limited for 6-24 h. Best results were obtained with retinol SLN incorporated in the oil-in-water (o/w) cream retarding drug expulsion. The penetration of the occlusion sensitive drug retinyl palmitate was even more influenced by SLN incorporation. Transepidermal water loss (TEWL) and the influence of drug free SLN on retinyl palmitate uptake exclude pronounced occlusive effects. Therefore enhanced retinyl palmitate uptake should derive from specific SLN effects and is not due to non-specific occlusive properties.

Solid lipid nanoparticles (SLN) based on binary mixtures of liquid and solid lipids: a (1)H-NMR study.

SLN with improved payloads and enhanced storage stability were investigated. Based on the experiences with solid lipid nanoparticles, a new type of solid lipid nanoparticle has been developed by incorporating triglyceride containing oils in the solid shell of the particle. The structure and mixing behaviour of these particles was characterised by DSC and (1)H-NMR. DSC yields information on the melting and crystallisation behaviour of the solid and liquid constituents of the particles. NMR is especially suited for the characterisation of the liquid oil domains inside the SLN. In this study a medium chain triglyceride oil was successfully incorporated in a matrix of a solid long chain glyceride (glyceryl behenate). The resulting particles were solid but the oil inside the particle remained in a liquid state. The relation between oil supplementation and melting point depression of glyceryl behenate proved to be linear. Mobility of the oil molecules inside the particles was considerably reduced compared to the emulsified oil. Moreover, two different chemical shifts for each of the lipid signals were observed indicating two different chemical environments. The experimental data is in line with a model describing uniform distribution of the oil molecules in the glyceryl behenate for low oil loads. However, at higher oil loads our data indicate the formation of oil clusters within the solid nanoparticle.

Characterisation of a novel solid lipid nanoparticle carrier system based on binary mixtures of liquid and solid lipids.

A drug carrier of colloidal lipid particles with improved payloads and enhanced storage stability was investigated. Based on the experiences with hard fats nanoparticles, a new type of solid lipid nanoparticles (SLN) has been developed by incorporating triglyceride containing oils in the solid core of said particle. The structure and mixing behaviour of these particles were characterised and practical implications on controlled release properties tested. Nanoparticles were characterised by their melting and recrystallisation behaviour as recorded by differential scanning calorimetry (DSC). Polymorphic form and bilayer arrangement were assigned by wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS). Size distribution and storage stability were investigated by laser diffractometry (LD). Release properties were studied by drug release model according to Franz. A medium chain triglyceride oil was incorporated successfully in a matrix of a solid long chain glyceride. The crystal order was greatly disturbed, however, the carrier remained solid. The oil inside the particle remained in a liquid state and induced a slight shift form the beta' polymorph to the beta(i) form. Long spacings varied within 0.1 nm with increasing oil loads. Nanoparticles with low oil concentrations showed sustained release properties. Improved drug load levels were encapsulated by lipid particles supplemented with oily constituents. Thus, the presented carrier adds additional benefits to the well-known opportunities of conventional SLN and is suited for topical use.

Scaling up feasibility of the production of solid lipid nanoparticles (SLN).

Solid lipid nanoparticles (SLN/Lipopearls) are widely discussed as colloidal drug carrier system. In contrast to polymeric systems, such as polylactic copolyol capsules, these systems show up with a good biocompatibility, if applied parenterally. The solid lipid matrices can be comprised of fats or waxes and allow protection of incorporated active ingredients against chemical and physical degradation. The SLN can either be produced by "hot homogenisation" of melted lipids at elevated temperatures or a "cold homogenization" process. This paper deals with production technologies for SLN formulations, based on non-ethoxylated fat components for topical application and high pressure homogenization (APV Deutschland GmbH, D-Lübeck). Based on the chosen fat components, a novel and easy manufacturing and scaling up method was developed to maintain chemical and physical integrity of encapsulated active and carrier.

Encapsulation of retinoids in solid lipid nanoparticles (SLN).

SLN have been suggested for a broad range of applications, such as intravenous injection, peroral, or dermal administration. The incorporation of the drug in the core of the SLN has to be ensured for these applications, but the inclusion of drugs in SLN is poorly understood. This study is a contribution to further describe the inclusion properties of colloidal lipids and to propose incorporation mechanisms. Besides the well known methods to investigate entrapment of actives in nanoparticles such as DSC or microscopy, the present study focussed on yet a different approach. Based on the different chemical stability of retinoids in water and in a lipid phase, a method to derive information on the distribution of the drug between SLN-lipid and the water phase was established. Comparing different lipids, glyceryl behenate gave superior entrapment compared to tripalmitate, cetyl palmitate and solid paraffin. Comparing three different drugs, entrapment increased with decreasing polarity of the molecule (tretinoin < retinol < retinyl palmitate). The encapsulation efficacy was successfully enhanced by formulating SLN from mixtures of liquid and solid lipids. These particles were solid and provided better protection of the sensitive drugs than an emulsion. X-ray investigations revealed that good encapsulation correlated with a low degree of crystallinity and lattice defects. With highly ordered crystals, as in the case of cetyl palmitate, drug expulsion from the carrier was more pronounced.

Medium scale production of solid lipid nanoparticles (SLN) by high pressure homogenization.

Solid lipid nanoparticles (SLN) were produced by high pressure homogenization using piston-gap homogenizers. Batch sizes varied between 40 ml and 50 l. Because of the different batch sizes, different homogenizer types were used, but the same functional principles were maintained, and the change from 40 ml to 50 l was not critical. With increasing batch sizes, the product quality in terms of particle size distribution and physical storage stability improved. Medium scale (30 l and 50 l) drug-free and drug-loaded SLN batches could be produced reproducibly and batch-to-batch uniformity was proven: within one batch particle sizes were homogeneous. This study revealed the influence of pressure and temperature for the hot homogenization technique A change of pressure between 300-500 bars induced only minor differences in particle size, but some influence of the heating temperature was found. More important than control of the heating process was the control of the cooling process of the final product. A too rapid cooling deteriorated the product quality: cooling with water of 18 degrees C proved to be the optimum cooling condition.