Physicochemical characterization of protein-loaded pectin-chitosan nanoparticles prepared by polyelectrolyte complexation.

Recent advances in nanotechnology applied to proteins are directed towards safer and simpler methods of preparation, using naturally occurring polymers such as alginate, pectin and chitosan. In this study, pectin-chitosan nanoparticles (NPs) were designed by the mild process of polyelectrolyte complexation, which occurs at room temperature without using sonication or organic solvents. NPs with a mean diameter between 300 and 400 nm and 45 to 86% protein association efficiency were obtained by varying the pectin:chitosan mass ratio and initial protein concentration. A prolonged release profile without burst effect of investigated ovalbumin from pectin-chitosan NPs was determined.

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.

An investigation into keratinolytic enzymes to enhance ungual drug delivery.

The topical therapy of nail diseases is limited by the low permeability of drugs through the nail plate. To increase drug penetration, the integrity of the nail plate must be compromised to a certain extent. We hypothesised that keratinolytic enzymes might decrease the barrier properties of the nail plate by hydrolysing the nail keratins, and thereby enhance ungual drug permeation. To determine enzyme action on nail plates, nail clippings were incubated at 35 degrees C, in the presence of keratinase at optimal pH for 48h, after which the nail plates were examined using scanning electron microscopy. It was found that the enzyme acted on the intercellular matrix which holds nail cells together, such that corneocytes on the dorsal surface separated from one another and 'lifted off' the nail plate. In addition, the surface of the corneocytes was corroded. Permeation studies using modified Franz diffusion cells and bovine hoof membranes as a model for the nail plate showed that the enzyme enhanced drug permeation through the hoof membrane. The permeability and partition coefficients, and the drug flux were found to be significantly increased in the presence of the enzyme. We can conclude that the enzyme, via its hydrolytic action on nail plate proteins, could increase ungual drug delivery.

Possible role of jasmonic acid in the regulation of floral induction, evocation and floral differentiation in Lemna minor L.

Jasmonic acid (JA) is implicated in a wide variety of developmental and physiological processes in plants. Here, we studied the effects of JA and the combination of JA and ethylenediamine-dio-hydroxyphenyl-acetic acid (EDDHA) on flowering in Lemna minor in axenical cultures. JA (0.475-47.5 nmol l(-1)) enhanced floral induction in L. minor under long-day (LD) conditions. Under the same conditions, at a concentration of 237.5 nmol l(-1), JA inhibited floral induction, and at a concentration of 475 nmol l(-1) it prevented floral induction. Under LD conditions with LD preculture, a combination of EDDHA (20,500 nmol l(-1)) and JA (47.5 nmol l(-1)) had a synergistic effect on the promotion of floral induction. Floral induction was enhanced to the greatest extent in experiments with LD precultures. Microscopic examination of microphotographs of histological sections showed that JA and, to an even greater extent, JA+EDDHA at optimal concentrations promote apical floral induction (evocation). Furthermore, JA, and to an even greater extent JA in combination with EDDHA in an optimal concentration, also promote flower differentiation, especially the development of stamens, as is evident from the microphotographs. The experimental results show that JA promotes floral induction in other species of Lemnaceae from various groups according to their photoperiodic response. The results support our hypothesis that, in addition to previously ascribed functions, JA may regulate floral induction, evocation and floral differentiation. Our hypothesis is supported also by the results obtained by quantitative determination of endogenous JA levels in L. minor at three growth stages. The levels of endogenous JA decreased from 389 ng JA g(-1) (fresh weight) of L. minor during the vegetative stage to 217 ng JA g(-1) during the evocation stage, and to 37.5 ng JA g(-1) during the flowering stage, which proves that JA is used for flowering.

Preparation and evaluation of nanosuspensions for enhancing the dissolution of poorly soluble drugs.

Poorly water-soluble compounds are difficult to develop as drug products using conventional formulation techniques and are frequently abandoned early in discovery. In the present study, the melt emulsification method traditionally used to prepare solid lipid nanoparticles was adapted to produce drug nanosuspensions. The method was evaluated in comparison with the well known solvent diffusion process for ibuprofen as a model drug. Control of the preparation variables (stabilizers, drug content, homogenization procedure and cooling conditions) allowed formation of nanosuspensions with diameters less than 100 nm. The major advantage of the melt emulsification method over the solvent diffusion method is the avoidance of organic solvents during production, although the mean particle size is slightly greater. The combination of Tween 80 and PVP K25 as stabilizers yields nanosuspensions with the smallest average particle size. The formulation of ibuprofen as a nanosuspension, either in the form of lyophilized powder or granules, was very successful in enhancing dissolution rate, more than 65% of the drug being dissolved in the first 10 min compared to less than 15% of the micronized drug. The increase in in vitro dissolution rate may favourably affect bioavailability and improve safety for the patient by decreasing gastric irritancy.

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.

The effect of hypoxia and low pH on the cytotoxicity of chlorambucil.

Studies with mouse tumors have shown that the effectiveness of certain chemotherapeutic agents can be enhanced if they are used in appropriate combination with an anti-hypertensive drug such as hydralazine. This results in reduced tumor blood flow with, among other things, a consequent decrease in oxygenation and increase in acidity in the tumor tissue. The purpose of the present work was to determine to what extent hypoxia and low pH are involved in the mechanism of this effect for chlorambucil. V79-WNRE cells were exposed to various drug concentrations under aerobic or hypoxic conditions, pH 6.4 or 7.4. Measurements of cell survival following 1 hr exposure at 37 degrees C showed that pH 6.4 produced a large potentiation of cell killing by chlorambucil (ER = 4 approx.); hypoxia, on the other hand, had little effect. The potentiation was shown to be greatest for pH values below 7.0. HPLC measurements of drug uptake were made since it was anticipated that chlorambucil, a weak acid, might tend to accumulate in cells under conditions of low extracellular pH. It was found that at an extracellular pH of 6.4 the ratio of the intracellular (Ci) and extracellular (Ce) drug concentrations was increased 4.5 and 3.6 fold for aerobic and hypoxic conditions, respectively. This probably explains most, if not all, of the cell killing potentiation observed at low pH.

Comparative rheological investigation of crude gastric mucin and natural gastric mucus.

Mucus is a viscoelastic gel covering the entire alimentary tract. It represents a pre-epithelial diffusion barrier influencing the drug absorption process. In this way the rheological properties of mucus play an important role. The purpose of our investigation was to evaluate and compare the rheological properties of rehydrated dried crude porcine gastric mucin (Sigma mucin) and isolated natural porcine gastric mucus. Two different rheological approaches were used: constant shear and oscillatory shear conditions. The results obtained under steady shear conditions show that the reduction of apparent viscosity is caused by time, shear rate and temperature. With increasing Sigma mucin concentration the rheological behaviour changes from time-independent systems fitting in with the Casson model to time-dependent systems fitting in with the Herschel-Bulkley rheological model. Measurements performed under oscillatory shear conditions clearly show how the mechanical response of different concentrated systems has changed from dispersions with prevailing plastic to prevailing elastic properties. Mechanical deformation and frequency response of natural gastric porcine mucus have shown a strong gel structure with elastic properties. Comparing both systems it can be concluded that after rehydration of dried crude gastric porcine mucin, a model mucus system with rheological properties equivalent to natural mucus cannot be obtained.

Improved skin oxygenation after benzyl nicotinate application in different carriers as measured by EPR oximetry in vivo.

The development of formulations, which increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with processes affected by the level of oxygen, e.g., rate of healing and efficiency of radiation oncology. In this study we have investigated the role of carriers on the efficacy of benzyl nicotinate (BN) action in skin after dermal application in different formulations by EPR oximetry in vivo. The time course of pO2 in the skin after application of rubefacient is followed directly for the first time. The results obtained proved the applicability of in vivo EPR oximetry as a sensitive method by which small alterations in pO2 can be detected. We have found that the type of vehicle significantly influences the time when BN starts to act, the duration of its action, and the maximal increase in pO2. The ranking of vehicle efficiency was: lipid nanoparticles in hydrophilic gel>liposomes in hydrophilic gel>hydrophilic gel>hydrophobic ointment>hydrophobic cream. Primarily the semi-solid vehicle determines the lag-time of action, but the maximal oxygen level is influenced decisively by the particulate carrier systems. BN effectiveness was dose dependent. 2.5% w/w concentration of BN appears to be the most appropriate for therapeutic application. After repeated application a successive increase of pO2 base line in skin and of the maximal pO2 was noticed.

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.

The cytotoxicity of melphalan and its relationship to pH, hypoxia and drug uptake.

In the present in vitro studies we examined the effect of hypoxia and acidic pH, two important consequences of reduced blood flow in vivo, on the cytotoxicity of melphalan treatment in Chinese hamster V79-WNRE and SiHa human tumor cells. Cells were exposed to various concentrations of melphalan for 1 hr at 37 degrees C under oxic or hypoxic conditions; pH 6.6 or 7.4, and cell survival was measured. The cytotoxicity of melphalan was potentiated by both low pH and hypoxia, in both cell lines. The overall potentiation, expressed as an enhancement ratio (ER), from both hypoxia and low pH was 3.5 in V79-WNRE and 2.9 in SiHa cells. The potentiation of cell killing produced by hypoxia alone (ERHyp) ranged from 1.4 to 1.9, and was greater in V79-WNRE than in SiHa cells. The potentiation from low pH (ERpH) was approximately 2 in both cell lines. HPLC analysis showed substantial intracellular accumulation of melphalan in both cell lines. Hypoxia and reduced pH further enhanced uptake of melphalan but this was not sufficient by itself to account for the increased potentiation of cytotoxicity observed under those conditions.

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.

Interactions of solid lipid nanoparticles with model membranes and leukocytes studied by EPR.

Solid lipid nanoparticles (SLN) are colloidal systems which have been proposed for several administration routes. Only limited data are available about the mechanism and rate of interaction of SLN with cells and tissues. The aim of our study was to investigate interactions of SLN with model membranes (liposomes) and cells (leukocytes). SLN dispersions composed of glyceryl tripalmitate, phosphatidylcholine, water, and poloxamer 188 or Tween 20 were prepared by the melt-emulsification process. Spin-labeled phosphatidylcholine (PC(10,3)) and the methylester of doxyl palmitic acid (MeFASL(10,3)) were incorporated into SLN as spin probes (SPs) in order to determine the rate and mechanism of cell interaction by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the exchange of SP between SLN and liposomes is much faster for MeFASL(10,3) than for PC(10,3), probably due to the smaller size of the former. In contrast to liposomes, in leukocytes no significant difference in the transfer rates of the two SP was observed after incubation, suggesting that there is an uptake of SLN to leukocytes (endocytosis) although simultaneous SP diffusion is not excluded. The interaction of SLN with leukocytes appears to depend significantly on the stabilizer used. Transfer of PC(10,3) from SLN coated with poloxamer 188 is much faster than from SLN coated with Tween 20.

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).

Influence of spin probe structure on its distribution in SLN dispersions.

Solid lipid nanoparticles (SLN) are drug carrier system composed of biodegradable substances, which are solid at room temperature. The physico-chemical properties and structure of the incorporated compounds can affect their partitioning in SLN dispersions. In this work the influence of lipophilicity and structure of different SP on its location in SLN were studied. By electron paramagnetic resonance (EPR) measurements it was found that lipophilic SP distribute between a solid glyceride core and a soft phospholipid layer, with the more polar part (piperidine ring or methylcarboxylic groups) oriented toward the water-lipid interface. The majority of SP is located in the phospholipid layer, but the portion in the solid lipid core increases with SP lipophilicity. The hydrophilic Tempol does not incorporate into SLN.

Viscosity prediction of lipophilic semisolid emulsion systems by neural network modelling.

Previously published data (Gasperlin et al., 1998) on viscoelastic behaviour of lipophilic semisolid emulsion systems and the prediction of their physical stability by neural network modelling are analysed in further detail. Most attention has been paid to viscosity, which with storage (G') and loss modulus (G"), is one of the most important rheological parameters influenced by structure. Complex dynamic viscosity (eta*) was measured by oscillatory rheometry. The viscosity dependence of the lipophilic semisolid emulsions on the ratio of the particular components was defined by the neural network (error back-propagation algorithm), linear and incomplete polynomial models of higher orders. Polynomial models were used to complement the neural network model and to determine the relationship between variables. Since the viscosity was expressed in the whole measured frequency range, modelling was more complex and indirect modelling was introduced. The determined models were tested and the results confirm their usefulness for the explanation and prediction of the rheological characteristics of emulsion systems. The trained and tested neural network model proved to be a highly effective and applicable tool for predicting the viscosity of a lipophilic semisolid emulsion system of given composition.

[The influence on some physicochemical parameters on drug release from PMMA salt hydrogels]. / Einfluss einiger physikochemischer Parameter auf die Wirkstofffreisetzung aus PMMA-Salz-Hydrogelen.

In vitro drug liberation results from PMMA (Eudispert)-salt hydrogels are explained from the aspect of polymer concentration, polymer molecular mass, viscosity as well of neutralisation grade and cation (Na+, K+, NH+4, ethanolamine, triethanolamine) of the polymer molecule and solubility properties of the drugs. Drug diffusion through hydrogel is regulated with the polymer network, in dependence of polymer nature and further with die mobility of water. The considered influences predominantly cover each other.

[Drug liberation from dermatica (author's transl)]. / Wirkstoff-Freisetzung aus Dermatica.

Investigated 0,1% ointments based on oleogels, hydrogels and O/W creams show differences by in-vitro evaluation of dexamethasone sodiumphosphate and dexamethasone release, which we have evaluated concerning the vehicle composition and its viscosity, the internal structure of systems and the drug properties. The results show dependence of drug availability on the vehicle gel structure order on the one, and on the drug activity on the other hand. The presence of a hydrocolloid (Eudispert-Na) in O/W creams improves the drug release.

The topography of electrospun nanofibers and its impact on the growth and mobility of keratinocytes.

Despite a lot of intensive research in the field of polymer nanofibers as wound-healing and tissue-regeneration materials, the behavior of cells in contact with nanofibers in vitro as well as in vivo is still not well understood. However, this knowledge is crucial for the design of nanofibrillar materials that are suitable for biomedical applications. Therefore, in this study, we present the preparation of poly(vinyl alcohol) (PVA) nanofibers from a physico-chemically characterized polymer solution by electrospinning together with a stabilization method to preserve the morphology of the nanofibers in aqueous conditions. An investigation of the effects of a nanofibrillar scaffold on the growth of human keratinocytes showed that randomly oriented PVA nanofibers delay the keratinocytes' adhesion but improve their strength, greatly alter their morphology, increase their metabolic activity, and limit their mobility. We have shown that due to the small interfiber pores, the whole cells are unable to penetrate into nanofibrillar network efficiently. However, flexible cell parts can penetrate into the nanofibrillar network, whereas the cell nuclei stay on the surface of electrospun scaffold. Additional reason for poor cell mobility is random orientation of nanofibers, which does not provide continuous routes for successful cell infiltration. Therefore, nanofibrillar support with nanosized interfiber pores could potentially be used to enable an efficient cell proliferation and accelerate surface-wound healing, but not for three-dimensional tissue regeneration. Finally, we showed that aligned nanofibers can successfully direct the migration and proliferation of cells, which is a crucial property of nanomaterials for the successful regeneration of tissues with a highly organized structure.

The use of a combination of different MR methods to study swelling of hydrophilic xanthan matrix tablets at different pHs.

Modified-release matrix tablets have been extensively used by the pharmaceutical industry as one of the most successful oral drug-delivery systems. The key element in drug release from hydrophilic matrix tablets is the gel layer that regulates the penetration of water and controls drug dissolution and diffusion. Magnetic resonance imaging (MRI) is a powerful, non-invasive technique that can help improve our understanding of the gel layer formed on swellable, polymer-matrix tablets, as well as the layer's properties and its influence on the drug release. The aim was to investigate the effects of pH and ionic strength on swelling and to study the influence of structural changes in xanthan gel on drug release. For this purpose a combination of different MRI methods for accurate determination of penetration, swelling and erosion fronts was used. The position of the penetration and swelling fronts were the same, independently of the different xanthan gel structures formed under different conditions of pH and ionic strength. The position of the erosion front, on the other hand, is strongly dependent on pH and ionic strength, as reflected in different thicknesses of the gel layers.