Julijana Kristl

Optimisation of floating matrix tablets and evaluation of their gastric residence time

The present investigation concerns the development of the floating matrix tablets, which after oral administration are designed to prolong the gastric residence time, increase the drug bioavailability and diminish the side effects of irritating drugs. The importance of the composition optimisation, the technological process development for the preparation of the floating tablets with a high dose of freely soluble drug and characterisation of those tablets (crushing force, floating properties in vitro and in vivo, drug release) was examined. Tablets containing hydroxypropyl methylcellulose (HPMC), drug and different additives were compressed. The investigation shows that tablet composition and mechanical strength have the greatest influence on the floating properties and drug release. With the incorporation of a gas-generating agent together with microcrystalline cellulose, besides optimum floating (floating lag time, 30 s; duration of floating, >8 h), the drug content was also increased. The drug release from those tablets was sufficiently sustained (more than 8 h) and non-Fickian transport of the drug from tablets was confirmed. Radiological evidence suggests that, that the formulated tablets did not adhere to the stomach mucus and that the mean gastric residence time was prolonged (>4 h).

The evidence for solid lipid nanoparticles mediated cell uptake of resveratrol.

The potential for colloidal carriers to increase drug bioavailability has spurred a renewed interest in their uptake mechanisms and movement within cells. Solid lipid nanoparticles (SLN) were used as a carrier for a promising chemopreventive drug, resveratrol (RSV). The effects of SLN, empty or loaded with RSV (SLN-RSV), on the internalization, growth, morphology, metabolic activity and genetic material of keratinocytes were compared to those of RSV in solution. Fluorescence images clearly showed that SLN with a size below 180 nm move promptly through the cell membrane, distribute throughout the cytosol, move successively among different cellular levels and localize in the perinuclear region without inducing cytotoxicity. RSV solubility, stability and intracellular delivery were all increased by loading into SLN. The release profile of RSV showed a biphasic pattern, reflecting its distribution in SLN. RSV in solution was slightly cytotoxic. That was prevented by loading it into solid lipid nanoparticles, which preserved cell morphology. The cytostatic effect of SLN-RSV was much more expressed than that of RSV in solution. Delivery of RSV by SLN contributes to effectiveness of RSV on decreasing cell proliferation, with potential benefits for prevention of skin cancer.

Toxicological Aspects of Long‐Term Treatment of Keratinocytes with ZnO and TiO2 Nanoparticles

Sunscreens containing ZnO and TiO(2) nanoparticles (NPs) are increasingly applied to skin over long time periods to reduce the risk of skin cancer. However, long-term toxicological studies of NPs are very sparse. The in vitro toxicity of ZnO and TiO(2) NPs on keratinocytes over short- and long-term applications is reported. The effects studied are intracellular formation of radicals, alterations in cell morphology, mitochondrial activity, and cell-cycle distribution. Cellular response depends on the type of NP, concentration, and exposure time. ZnO NPs have more pronounced adverse effects on keratinocytes than TiO(2). TiO(2) has no effect on cell viability up to 100 μg mL(-1), whereas ZnO reduces viability above 15 μg mL(-1) after short-term exposure. Prolonged exposure to ZnO NPs at 10 μg mL(-1) results in decreased mitochondrial activity, loss of normal cell morphology, and disturbances in cell-cycle distribution. From this point of view TiO(2) has no harmful effect. More nanotubular intercellular structures are observed in keratinocytes exposed to either type of NP than in untreated cells. This observation may indicate cellular transformation from normal to tumor cells due to NP treatment. Transmission electron microscopy images show NPs in vesicles within the cell cytoplasm, particularly in early and late endosomes and amphisomes. Contrary to insoluble TiO(2), partially soluble ZnO stimulates generation of reactive oxygen species to swamp the cell redox defense system thus initiating the death processes, seen also in cell-cycle distribution and fluorescence imaging. Long-term exposure to NPs has adverse effects on human keratinocytes in vitro, which indicates a potential health risk.

Investigation of polymeric nanoparticles as carriers of enalaprilat for oral administration.

Enalaprilat is a typical angiotensin-converting enzyme inhibitor and is very poorly absorbed from the gastrointestinal tract. The aim of this study was to design and characterize poly-(lactide-co-glycolide) (PLGA) and polymethylmethacrylate (PMMA) nanoparticles containing enalaprilat and to evaluate the potential of these colloidal carriers for the transport of drugs through the intestinal mucosa. Nanoparticle dispersions were prepared by the emulsification-diffusion method and characterized according to particle size, zeta potential, entrapment efficiency and physical stability. Effective permeabilities through rat jejunum of enalaprilat in solution and in enalaprilat-loaded nanoparticles were compared using side-by-side diffusion chambers. The solubility of enalaprilat is very low in many acceptable organic solvents, but in benzyl alcohol is sufficient to enable the production of nanoparticles by the emulsification-diffusion process. The diameters of drug-loaded PMMA and PLGA nanoparticles were 297 and 204 nm, respectively. The concentration of the stabilizer polyvinyl alcohol (PVA) in dispersion has an influence on particle size but not on drug entrapment. The type of polymer has a decisive influence on drug content--7 and 13% for PMMA and PLGA nanoparticles, respectively. In vitro release studies show a biphasic release of enalaprilat from nanoparticle dispersions-fast in the first step and very slow in the second. The apparent permeability coefficient across rat jejunum of enalaprilat entrapped in PLGA nanoparticles is not significantly improved compared with enalaprilat in solution.

Effect of resveratrol incorporated in liposomes on proliferation and UV-B protection of cells.

The possibility of improving the efficacy of resveratrol, a polyphenol with strong antioxidant and free-radical scavenging properties, on cell proliferation and photoprotection by liposomal incorporation was investigated. Oligolamellar vesicles of different lipid compositions, loaded with resveratrol, were prepared and characterized by evaluating size, zeta potential, incorporation efficiency, electron microscopy and stability over 60 days. The effect of free and liposomal resveratrol on the viability of HEK 293 cells and their photoprotection after UV-B irradiation was assessed by the MTS method. Resveratrol decreased the cell viability at 100microM concentration, while at 10microM increased cell proliferation and also achieved the most effective photoprotection. Photomicrographs of the treated cells from inverted light and fluorescence microscopy demonstrated resveratrol effectiveness at 10microM, as well as its toxicity at higher concentrations, based on changes in cell shape, detachment and apoptotic features. Interestingly, liposomes prevented the cytotoxicity of resveratrol at high concentrations, even at 100microM, avoiding its immediate and massive intracellular distribution, and increased the ability of resveratrol to stimulate the proliferation of the cells and their ability to survive under stress conditions caused by UV-B light.

Advantages of celecoxib nanosuspension formulation and transformation into tablets.

Drugs with low aqueous solubility and high permeability (BCS class II) present a high proportion of all drugs. This study examines the critical issues regarding engineering of a nanosuspension tailored to increase drug dissolution rate and its transformation into dry powder suitable for tabletting. Nanosuspensions of celecoxib, a selective COX-2 inhibitor with low water solubility, were produced by the emulsion-diffusion method using three different stabilizers (Tween) 80, PVP K-30 and SDS) and characterized by particle size analysis, dissolution testing, scanning electron microscopy imaging, differential scanning calorimetry and X-ray powder diffraction. Spray-dried nanosuspension was blended with microcrystalline cellulose, and compressed to tablets, and their tensile strength, porosity and elastic recovery of tablets were investigated. The selection of solvent and stabilizers is critical, firstly to achieve controlled crystallization and size, and secondly to increase the wettability of the hydrophobic drug. The crystalline nano-sized celecoxib alone or in tablets showed a dramatic increase of dissolution rate and extent compared to micronized. SEM images showed that the nanoparticle morphology was influenced by the choice of stabilizers. Celecoxib nanosuspension stabilized with PVP K-30 and SDS showed advantages over Tween 80 due to sticking of the dried product and unexpected changes observed on DSC curves. Markedly lower compaction forces are needed for nano-sized compared to micro-sized celecoxib to produce tablets of equal tensile strength.

Liposomes as a topical delivery system: the role of size on transport studied by the EPR imaging method

The relative contribution of the liposome size, lamellarity, composition and charge to the transport into the skin of drug, which was applied entrapped in liposomes is a subject of some controversy. In this study the influence of liposome size on the transport of hydrophilic substance was investigated. For this purpose liposomes composed of dipalmitoylphosphatidylcholine (DPPC), or non-hydrogenated soya lecithin (NSL) or hydrogenated soya lecithin (HSL), all in combination with 30% cholesterol, as well as of two types of niosomes: from glyceryl distearate or PEG stearate in combination with 45% of cholesterol and 10% of lipoaminosalt were prepared and their physical characteristics (size, polydispersity index, zeta potential, entrapped volume) were determined. Their size was varied by extrusion and by sonication. The transport of the entrapped spin labeled hydrophilic compounds into the skin was measured by electron paramagnetic resonance imaging methods. No significant transport into the deeper skin layers (more than 100 microm deep) was observed for NSL liposomes, irrespective of vesicle size. For all other vesicular systems some transport into the deeper skin layers was observed, which did not depend on vesicle size, significantly until the vesicle diameter of approximately 200 nm was reached. However, for small vesicles (with diameter less than 200 nm) the transport is significantly decreased. We have proven that small vesicles are not stable and disintegrate immediately in contact with other surfaces. As a consequence, they lose an important influence on the topical delivery of the entrapped hydrophilic substances.

Effect of colloidal carriers on ascorbyl palmitate stability

Active compounds can be protected against degradation by incorporation into colloidal carrier systems. The stabilizing effect of carrier systems for ascorbyl palmitate (AP) was investigated using microemulsions (ME), liposomes and solid lipid nanoparticles (SLN). Analysis of chemical stability by HPLC showed that AP is most resistant against oxidation in non-hydrogenated soybean lecithin liposomes, followed by SLN, w/o and o/w ME, and hydrogenated soybean lecithin liposomes. The molecular environment of the AP-like nitroxide probe (C(16)-Tempo) in colloidal carriers was characterized using electron paramagnetic resonance (EPR) spectroscopy. We have found that the nitroxide groups are located in environments with different polarity and mobility. The hydrophilic part of AP is the reactive moiety, and high stability is obtained in systems in which this part is exposed to a less polar environment. Additionally, the determined accessibility of nitroxide groups to reduction correlated well with the chemical stability of AP. It is more deeply immersed in the interface when entrapped in a liquid-state carrier than when applied in gel-state particles. Encapsulation of AP in SLN core leads to greater stability. We conclude that the location of the sensitive group of the drug-molecule in a carrier system is crucial for its stability.

Improvements of cellular stress response on resveratrol in liposomes

Resveratrol (RSV) has proven potential in prophylaxis and treatment of various disorders mediated by free radicals and oxidative stress. RSV solubility, stability, and cytotoxicity must be regulated for satisfactory bioavailability. Here, RSV was loaded into liposomes, characterized by PCS and TEM and evaluated on HEK293 cell line by metabolic activity assay, electron paramagnetic resonance, and fluorescence microscopy. RSV at 10 microM induced changes in cell metabolic activity and significantly improved antioxidative capacity. At 100 microM it showed concentration-dependent cytotoxicity. Oligolamellar liposomes with mean diameter 84 nm, polydispersity index 0.2, and zeta potential -40 mV showed high entrapment of RSV and rapid cellular internalization. Cell stress caused by UV-B irradiation diminished cell metabolic activity by 50%. RSV loaded into them showed no cytotoxicity at 100 microM and stimulated cellular metabolic and antioxidant activity levels to eliminate the harmful effect of the stress. Localization of RSV within liposomal bilayer is crucial for stimulation of cell-defense system, prevention of RSV cytotoxicity, and its long-term stability. In summary, evidence of different metabolic activity using free RSV and LIP-RSV is presented indicating that liposome-mediated uptake of RSV is more effective for improvement of the cell-stress response.

Critical attributes of nanofibers: preparation, drug loading, and tissue regeneration.

The electrospinning technique for nanofiber production has opened new and interesting opportunities for tissue regeneration and treatment, because they allow biomimetic supports for cell growth to be designed and enable simultaneous delivery of diverse drugs in a controlled manner. In this review the process of electrospinning itself and the parameters affecting the electrospinning outcome are presented in detail. Critical issues related to nanofiber composition and drug loading and analytical tools for characterization and quality assurance of electrospun nanofibers are described. Recent findings about the response of cells grown on nanofibrillar supports, including cell adhesion, morphology, proliferation, and mobility, are also introduced. This review summarizes the progress that has been made in recent years on nanofibers for biomedical use and highlights the major challenges that still remain to be solved.