Comparison of four different fuller's earth formulations in skin decontamination.

Industrial accidents, wars and terrorist threats are potential sources of skin contamination by highly toxic chemical warfare agents and manufacturing compounds. We have compared the time-dependent adsorption capacity and decontamination efficiency of fuller's earth (FE) for four different formulations for the molecular tracer, 4-cyanophenol (4-CP), in vitro and ex vivo using water decontamination as standard. The adsorption capacity of FE was assessed in vitro for 4-CP aqueous solutions whereas decontamination efficiency was investigated ex vivo by tracking porcine skin 4-CP content using attenuated total reflectance Fourier transform infrared spectroscopy. Decontamination was performed on short time, exposed porcine skin to 4-CP by application of FE: (1) as free powder; (2) loaded on adhesive tape; (3) on powdered glove; or (4) in suspension. Removal rate of 4-CP from aqueous solutions correlates with the amount of FE and its contact time. Decontamination efficiency estimated by the percentage of 4-CP recovery from contaminated porcine skin, achieved 54% with water, ranged between ~60 and 70% with dry FE and reached ~90% with FE suspension. Successful decontamination of the FE suspension, enabling a dramatic reduction of skin contamination after a brief exposure scenario, appears to be rapid, reliable and should be formulated in a new device ready to use for self-application.

The influence of microemulsion structure on their skin irritation and phototoxicity potential.

The purpose of this study was to examine skin irritation and phototoxicity potentials of several microemulsions (ME), all comprising approximately the same percentage of surfactant mixture, but varying oil/water content and consequently inner structure being either droplet-like (o/w ME, o/w ME carbomer, w/o ME and w/o ME white wax) or lamellar (gel-like ME). Two different in vitro methods were used: MTT assay (performed either on reconstructed human epidermis (RHE) or NCTC 2544 cells) and pig ear test. Neither assay revealed the difference among ME with droplet-like structure. Then again, pig ear test and MTT assay performed on RHE indicated that gel-like ME is more irritant compared to other tested ME, whereas no difference among formulations were observed by MTT assay on NCTC 2544 cells. The reasonable explanation is destruction and consequently uniform structure of ME upon dilution that is inevitable for testing on cell cultures. The results of phototoxicity test again indicated the increased potential of gel-like ME to cause adverse effects on skin. It can be concluded that for ME consisting of the same amount of identical surfactants but having different structure the latter represent a crucial factor that determines their dermal toxicity.

Microemulsion system for topical delivery of thai mango seed kernel extract: development, physicochemical characterisation and ex vivo skin permeation studies.

A microemulsion system containing Thai mango seed kernel extract (MSKE, cultivar "Fahlun") was developed and characterised for the purpose of topical skin delivery. The MSKE-loaded microemulsions were prepared by using the spontaneous emulsification method. Isopropyl myristate (IPM) was selected as the oil phase. A polyoxyethylene sorbitan monooleate and sorbitan monododecanoate (1:1, w/w) system was used as the surfactant phase; an aqueous mixture of different cosurfactants (absolute ethanol, 96.3% v/v ethanol, 1-propanol, 2-propanol or 1,2-propanediol) at a weight ratio of 1:1 was used as the aqueous phase. Among the cosurfactants studied, the 1-propanol aqueous mixture had the largest microemulsion region (48.93%) in the pseudo-ternary phase diagram. Microemulsions containing 1% MSKE demonstrated good physicochemical stability during a six-month study period at 25 ± 2 °C/60% ± 5% RH. The ex vivo skin permeation study demonstrated that the microemulsions exhibited a potent skin enhancement effect allowing MSKE to penetrate skin layers up to 60-fold higher compared with the control. Neither skin irritation nor skin corrosion was observed in ex vivo studies. The present study revealed that IPM-based microemulsion systems may be promising carriers to enhance skin penetration and delivering MSKE for topical treatment.

Chlorhexidine salt-loaded polyurethane orthodontic chains: in vitro release and antibacterial activity studies.

The widespread use of indwelling medical devices has enormously increased the interest in materials incorporating antibiotics and antimicrobial agents as a means to prevent dangerous device-related infections. Recently, chlorhexidine-loaded polyurethane has been proposed as a material suitable for the production of devices which are able to resist microbial contamination. The aim of the present study was to characterize the in vitro release of chlorhexidine from new polymeric orthodontic chains realized with polyurethane loaded with two different chlorhexidine salts: chlorhexidine diacetate or chlorhexidine digluconate. The orthodontic chains constituted of three layers: a middle polyurethane layer loaded with chlorhexidine salt inserted between two layers of unloaded polymer. In vitro release of chlorhexidine diacetate and digluconate from orthodontic chains loaded with 10% or 20% (w/w) chlorhexidine salt was sustained for 42 days and followed Fickian diffusion. The drug diffusion through the polyurethane was found to be dependent not only on chlorhexidine loading, but also on the type of chlorhexidine salt. The antibacterial activity of 0.2% (w/w) chlorhexidine diacetate-loaded orthodontic chain was successfully tested towards clinically isolated biofilm forming ica-positive Staphylococcus epidermidis via agar diffusion test. In conclusion, the chlorhexidine salt-loaded chains could provide an innovative approach in the prevention of oral infections related to the use of orthodontic devices.

Preparation and evaluation of nanoparticles for directed tissue engineering.

Herein we describe the preparation of a nanoparticulate system formed from an RGD-functionalized chitosan derivative by complexation with chondroitin sulfate. These bioactive complexes were developed to promote wound healing by inducing adhesion and subsequently migration of skin cells. The particles were characterized for their size, surface charge, stability and shape. Briefly, the nanoparticles were found to be stable up to 7 days in water at a diameter of 150-200 nm and a positive charge of 20 mV. In physiological media the particles swell significantly but remain intact. Tested in an in vitro cell model of human dermal fibroblasts, the particles were shown to promote cell adhesion and induce spreading in human dermal fibroblasts. The mean surface area per cell was found to be increased by three-fold (n=3 assays, p<0.01), for the cells plated on particles exposing RGD-peptides when compared to cells on control particles. This indicates a stimulation of the cells due to the exposure of the bioactive RGD-moieties and an enhanced cell-biomaterial interaction. Using nanoparticles is a novel approach to direct cellular behavior with numerous possible applications in tissue engineering such as substrate for dermal and epithelial cells, injectable suspensions or as building blocks to form scaffolds.

In vitro evaluation of an RGD-functionalized chitosan derivative for enhanced cell adhesion.

Tissue repair is a spontaneous process that is initiated on wounding. However, if this complex mechanism is impaired or not sufficient the use of biomaterials might increase the chance of successful healing. In this view, an RGD-functionalized polymer was developed to promote dermal healing. A water-soluble chitosan derivative, carboxymethyl-trimethylchitosan (CM-TM-chitosan) was synthesized and GRGDS-moieties were grafted to the backbone at a concentration of 59 nmol/mg polymer to increase cell-biomaterial interaction. Tested in vitro with cultured human dermal fibroblasts, the developed polymer showed good biocompatibility and the initial adhesion was increased by 3-5 times due to the GRGDS-moieties. Moreover, cell spreading was specific to the interaction with GRGDS, giving a 12-fold increase of cells showing a fully spread morphology within 30 min. Overall, CM-TM-chitosan conjugated with GRGDS-peptides may prove useful as a biomaterial in wound healing.

Transport of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) in human respiratory epithelial cells.

Organic cation/carnitine transporters (OCT/N) mediate uptake of positively charged molecules. Their role in lung epithelium; however, is not well understood. OCT/N expression and activity was studied in cell lines of human alveolar (A549), bronchial (16HBE14o- and Calu-3) and intestinal (Caco-2) epithelium. Protein levels were largely comparable for all OCT/Ns in the respiratory epithelial cell lines studied; however, OCT2 was exclusively observed in A549 cells. OCT1 and -2 were present at significantly higher levels in Caco-2 cells, compared with the pulmonary epithelial cell types. OCTN1 and -2 were also more abundant in Caco-2. Only OCT3 was expressed evenly across all cell lines investigated. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was dependent on concentration, temperature, membrane potential and pH. In 16HBE14o-, Calu-3 and Caco-2 monolayers, substrate saturation of ASP(+) uptake was not reached. Alveolar A549 cells showed saturable ASP(+) uptake via two transporter sites with K(m) values of 12.5 ± 4.0 µM and 456.9 ± 164.5 µM, respectively. This uptake was sensitive to organic cations, but insensitive to carnitine and lysine. We conclude that uptake of organic cations is facilitated by distinct pathways in different regions of lung mucosa. Luminally localised OCT2 appears to be exclusively involved in the alveolar epithelium, whereas basolateral localised OCT3 might play a role in alveolar as well as in bronchial epithelial cells.

Solid lipid nanoparticles suspension versus commercial solutions for dermal delivery of minoxidil.

Solid lipid nanoparticles have been reported as possible carrier for skin drug delivery. Solid lipid nanoparticles are produced from biocompatible and biodegradable lipids. Solid lipid nanoparticles made of semi-synthetic triglycerides stabilized with a mixture of polysorbate and sorbitan oleate were loaded with 5% of minoxidil. The prepared systems were characterized for particle size, pH and drug content. Ex vivo skin penetration studies were performed using Franz-type glass diffusion cells and pig ear skin. Ex vivo skin corrosion studies were realized with a method derived from the Corrositex(®) test. Solid lipid nanoparticles suspensions were compared to commercial solutions in terms of skin penetration and skin corrosion. Solid lipid nanoparticles suspensions have been shown as efficient as commercial solutions for skin penetration; and were non-corrosive while commercial solutions presented a corrosive potential. Solid lipid nanoparticles suspensions would constitute a promising formulation for hair loss treatment.

Development and in vitro assay of oxidative stress modifying formulations for wound healing promotion.

Often presented as metabolism byproducts, reactive oxygen species are linked to detrimental effects such as chronic wound, mutagenesis, cancer and skin ageing. However, recent in vitro and in vivo observations suggest that ROS, and mainly hydrogen peroxide, interfere with cell signaling acting like second messenger and inducing adaptive responses. This is particularly observed in skin wound healing where cells are exposed to H2O2 following injury. In this study, we developed and characterized an innovative formulation producing H2O2 at low concentrations, in order to mimic physiological inflammation phase. Then, this pro-oxidative formulation (CAM-GOx) was assayed in vitro on keratinocytes cell culture, compared to the blank formulation (CAM) and the anti-oxidative formulation (CAM-CAT) to assess whether oxidative stress was implied or not in cellular responses.

Compartmentalization of the human stratum corneum by persistent tight junction-like structures.

Several tight junction (TJ) proteins were detected in the living layers of adult human epidermis, and TJ-like membrane ridges were observed at the top of the stratum granulosum (SG) in freeze-fracture studies. We applied standard and immunoelectron microscopy to look for TJ-derived structures in the stratum corneum (SC) of human adult epidermis and in cornified envelopes purified from the plantar SC. Besides confirming claudin-1 labelling in the proximity of SG desmosomes, we also observed immunolocalization near corneodesmosomes in the lower SC. In addition, TJ proteins were consistently detected in the purified cornified envelopes. Lateral but not horizontal walls of the corneocytes showed frequent points of molecular fusion between lipid envelopes. These structural associations were very frequently localized at the top of the lateral corneocyte membranes, thus sealing the extremities of lateral intercorneocyte spaces. We propose that TJ-like structures persist in the SC and contribute to the reinforcement of lateral contacts and to the formation of membrane interdigitations between corneocytes. Their presence could contribute to subdivision of the extracellular spaces of SC into consecutive individualized compartments. Intercellular lipids, enzymes and other (glyco)protein content could thus evolve in the keratinized epidermal layer at different paces, as preprogrammed in the underlying living cells and influenced by the environment, e.g. humidity. Such situation might explain differences in the degradation rates between the 'peripheral' and the 'non-peripheral' corneodesmosomes observed during physiological desquamation, as previously suggested by us and others.

Reverse transcription polymerase chain reaction (RT-PCR) analysis of proteolytic enzymes in cultures of human respiratory epithelial cells.

BACKGROUND: Pancreatic proteolytic digestive enzymes are a major extracellular barrier to the sucessful systemic delivery of biopharmaceuticals via the oral route, whereas in health in the lungs these powerful proteases are virtually absent from the extracellular fluids. Despite this, the absorption of some (but not all) natural peptides and proteins from the lungs may be poor, and one has to acknowledge that information on the activity and spatial distribution of proteolytic enzymes in the human lung is scarce. Here, we investigated expression patterns of a series of proteolytic enzymes in several human respiratory cell types on mRNA level in an attempt to better understand the fate of inhaled biopharmaceuticals. METHODS: The mRNA expression of proteolytic enzymes (i.e., carboxypeptidases: CPA1, CPA2, CPB, CPM; gamma-glutamyltransferases: GGT1, GGT2; angiotensin-converting enzymes: ACE, ACE2; aminopeptidases: APA, APB, APN, APP1, APP2, APP3; endopeptidases: 24.11 (neprilysin), 24.15 (thimet oligopeptidase), 24.18 (meprin A); enteropeptidase; trypsin 1, trypsin 2; neutrophilic elastase; dipeptidyl peptidase 4; gamma-glutamylhydrolase) was investigated by semiquantitative RT-PCR in human bronchial (hBEpC, Calu-3, 16HBE14o-) and alveolar (A549) epithelial cells, respectively. Gastrointestinal Caco-2 cells were used as comparison. RESULTS: Obvious differences were observed in proteinases' expression pattern between the investigated cell types. Although considered to be of bronchial epithelial phenotype, neither Calu-3 nor 16HBE14o- cells matched the mRNA expression pattern of hBEpC in primary culture. Of all investigated cell lines, Caco-2 expresses the highest number of proteases and peptidases. CONCLUSIONS: Although mRNA expression does not necessarily signify enzyme functionality, our results provide the first comprehensive analysis of peptidase and protease expression and distribution in human lung epithelial cells and are the basis for further investigations.

Effects of glycerol on human skin damaged by acute sodium lauryl sulphate treatment.

Glycerol, widely used as humectant, is known to protect against irritants and to accelerate recovery of irritated skin. However, most studies were done with topical formulations (i.e. emulsions) containing glycerol in relatively high amounts, preventing drawing conclusions from direct effects. In this study, acute chemical irritations were performed on the forearm with application of a 10% sodium lauryl sulphate (SLS) aqueous solution under occlusion for 3 h. Then, glycerol aqueous solutions from 1 to 10% were applied under occlusion for 3 h. After elimination of moist excess consecutive to occlusive condition, in ambient air for 15 and 30 min, skin barrier function was investigated by dual measurement of skin hydration and transepidermal water loss (TEWL). Treatments with SLS solution under occlusion significantly increased TEWL and decreased skin hydration as assessed by capacitance measurements. The SLS irritant property was raised by the occlusion and the water barrier function as well as water content appeared impaired. Recovery with glycerol at low doses was remarkable through a mechanism that implies its hygroscopic properties and which is saturable. This precocious effect acts through skin rehydration by enhancing water-holding capacity of stratum corneum that would facilitate the late physiological repair of impaired skin barrier. Thus, glycerol appears to substitute for natural moisturizing factors that have been washed out by the detergent action of SLS, enhancing skin hydration but without restoring skin barrier function as depicted by TEWL values that remained high. Thus, irritant contact dermatitis treated with glycerol application compensate for skin dehydration, favouring physiological process to restore water barrier function of the impaired skin. Empirical use of glycerol added topical formulations onto detergent altered skin was substantiated in the present physicochemical approach.

Characterization of a polyurethane-based controlled release system for local delivery of chlorhexidine diacetate.

Conventional formulations of chlorhexidine usually provide short-term efficiency, requiring repeated applications to maintain antibacterial activity. Therefore, appropriate release system of chlorhexidine controlling local drug delivery would reduce the number of applications and enhance patient compliance. The aim of this study was to develop a controlled release system based on medical polyurethane for the local delivery of chlorhexidine diacetate (CDA). CDA-loaded polyurethane films (CDA-Films) and CDA-loaded polyurethane sandwiches (CDA-Sandwiches) were obtained by casting and solvent evaporation. The physico-chemical aspects of CDA-loaded polyurethane systems were investigated, and the crystalline state of CDA in the polymeric system was highlighted. CDA-Films exhibited appropriate mechanical properties for further applications. Drug release was measured in two different media: (i) distilled water and (ii) physiological saline solution to mimic in vivo conditions. Drug release studies were performed up to 11days on CDA-Films and 29days for CDA-Sandwiches. Release of CDA depended on drug loading and the structure of the system. In particular, release of CDA from the sandwich system followed zero-order kinetic. The release rate was significantly lower in physiological solution. Antibacterial studies were carried out on CDA-Films against Staphylococcus aureus and Staphylococcus epidermidis showing 35days persisting antibacterial activity. In conclusion, the polyurethane-based system developed in this study is potentially useful as a local delivery system for CDA and could be used not only in surgery but also in dental and clinical applications.

Self-organization of synthetic cholesteryl oligoethyleneglycol glycosides in water.

Lectin-sugar recognition systems are of interest in the pharmaceutical field, especially for the development of drug carriers, tailored for selective delivery. This paper deals with the anhydrous and aqueous self-organization properties of a synthetic cholesteryl oligoethyleneglycol glycoside with the aim of their incorporation in liposomes. Successive phases (lamellar, R3m, Im3m, micelles) have been described depending on water content and temperature. As a result of the presence of sugar residues and their hydration ability, this glycolipid shows a large range of packing parameter with increasing water content. However, because of oligoethyleneglycol spacer, a slight dehydration has been observed with increasing temperature from 20 to 60 degrees C.

Glycosylated liposomes against Helicobacter pylori: behavior in acidic conditions.

Helicobacter pylori was isolated in 1982 and confirmed as a gastric pathogenic agent at the end of the 1980s. The present work deals with liposomes formulations in which are incorporated cholesteryl tetraethylene glycol oside as model ligands for H. pylori adhesins. This study is devoted to the behavior of liposomes in gastric conditions. The glycosylated vesicles are stable and the pH of the internal aqueous compartment remains close to 4 even through more acidic conditions are imposed to the external phase (pH 1.2-2). Such a pH gradient depends essentially on the nature of phospholipids used and is not extensively affected by the incorporation of the targeting agent. These aspects are particularly important to the development of liposome formulations against H. pylori, bacteria sensitive to antibiotics which are unstable in very acidic conditions.

Temperature-sensitive microemulsion gel: an effective topical delivery system for simultaneous delivery of vitamins C and E.

Microemulsions (ME)--nanostructured systems composed of water, oil, and surfactants--have frequently been used in attempts to increase cutaneous drug delivery. The primary objective addressed in this work has been the development of temperature-sensitive microemulsion gel (called gel-like ME), as an effective and safe delivery system suitable for simultaneous topical application of a hydrophilic vitamin C and a lipophilic vitamin E. By changing water content of liquid o/w ME (o/w ME), a gel-like ME with temperature-sensitive rheological properties was formed. The temperature-driven changes in its microstructure were confirmed by rotational rheometry, viscosity measurements, and droplet size determination. The release studies have shown that the vitamins' release at skin temperature from gel-like ME were comparable to those from o/w ME and were much faster and more complete than from o/w ME conventionally thickened with polymer (o/w ME carbomer). According to effectiveness in skin delivery of both vitamins, o/w ME was found the most appropriate, followed by gel-like ME and by o/w ME carbomer, indicating that no simple correlation between vitamins release and skin absorption could be found. The cytotoxicity studies revealed good cell viability after exposure to ME and confirmed all tested microemulsions as nonirritant.

Simultaneous absorption of vitamins C and E from topical microemulsions using reconstructed human epidermis as a skin model.

Antioxidants provide the mainstay for skin protection against free radical damage. The structure of microemulsions (ME), colloidal thermodynamically stable dispersions of water, oil and surfactant, allows the incorporation of both lipophilic (vitamin E) and hydrophilic (vitamin C) antioxidants in the same system. The objective of this work was to investigate the potential of non-thickened (o/w, w/o and gel-like) and thickened (with colloidal silica) ME as carriers for the two vitamins using reconstructed human epidermis (RHE). The amounts of these vitamins accumulated in and permeated across the RHE were determined, together with factors affecting skin deposition and permeation. Notable differences were observed between formulations. The absorption of vitamins C and E in RHE layers was in general enhanced by ME compared to solutions. The incorporation of vitamins in the outer phase of ME resulted in greater absorption than that when vitamins were in the inner phase. The location of the antioxidants in the ME and affinity for the vehicle appear to be crucial in the case of non-thickened ME. Addition of thickener enhanced the deposition of vitamins E and C in the RHE. By varying the composition of ME, RHE absorption of the two vitamins can be significantly modulated.

Effects of physical and chemical treatments upon biophysical properties and micro-relief of human skin.

The aim of the present study was to determine the attendant effects of physical (tape-stripping) and chemical (three commercial hydrating formulations) treatments upon biophysical and micro-relief properties of human skin. In the first set of experiment, the effects of tape-stripping onto human stratum corneum (SC) biophysical and micro-relief properties were assessed in nine volunteers. Transepidermal water loss (TEWL), skin hydration and micro-relief parameters (including total length of the lines in mm per mm(2); total surface in %; roughness of the skin measured in gray level (Ra); maximum profile valley (Rv) depth; maximum profile peak height (Rp); maximum height (Rt), peak density (Pc) and coefficient of anisotropy) were determined by using SkinEvidence Pro after subsequent tape-stripping of SC. The relevance of roughness determination as gray level by SkinEvidence Pro was confirmed by using surface roughness standards. In the second set of experiment, the effectiveness of three commercial hydrating formulations onto human SC biophysical parameters and micro-relief properties were assessed in six volunteers. TEWL, hydration and micro-relief parameters were assessed onto pre-treated acetone skin and then treated by three commercial hydrating formulations after 2, 4 and 6 h skin exposure. A linear relation between hydration and cutaneous parameters (total length of the lines, Ra and Rp) as function of SC removed was shown. Skin barrier properties evaluated by TEWL measurements, were not modified by topical formulations. However, skin treated by topical formulations showed slightly higher hydration than the one determined in control group, while micro-relief parameters were not modified. In this study was showed that biophysical and micro-relief parameters were closely related in tape-stripping experiment. Efficiency of topical formulations was suggested upon skin hydration but not onto skin micro-relief and barrier function recovering. From both experiments, it appears that different mechanisms relating to skin hydration and potential modification of cutaneous micro-relief were suggested.

Pre-formulation of liposomes against Helicobacter pylori: characterization and interaction with the bacteria.

This paper deals with the formulation of targeted liposome against Helicobacter pylori. We describe the characterization of liposomes loaded with antimicrobial agents (ampicillin and metronidazole) and the quantification of the interactions between such formulations and bacteria. If the encapsulation rate of ampicillin seems not strongly affected by the change of phospholipidic composition, the encapsulation of metronidazole drastically decreased in epikuron 170 liposomes compared to DPPC ones. Furthermore, as observed with X-ray diffraction measurements, the presence of metronidazole results in the disorganisation of the phospholipid bilayers. Concerning the liposome-bacteria interactions, it has been observed that the incorporation of fucosyled glycolipids in the vesicle membrane leads to liposomes that are able to interact with the bacteria either in their spiral or in their coccoid forms. Since coccoid forms are occasionally found in vivo, their recognition by the liposomes we have formulated seems promising in the fight against Helicobacter pylori.

Study on the hydatid cyst membrane: permeation of model molecules and interactions with drug-loaded nanoparticles.

The success of the chemotherapeutic treatment of hydatid disease is based upon the drug ability to operate on the germinal layer and on the protoscolices of the hydatid cyst interior at adequate concentrations for sufficient periods. The goal of this study was to evaluate the ability of the drug diffusion through the cyst membrane from sheep hydatid cysts and the increase of drug concentration in the cyst environment. In the first part of this work, the permeation behaviour through the hydatid cyst membrane was studied with five model molecules, having different molecular descriptors (logP, molecular weight, polar surface area ...) onto static Franz glass diffusion cells. A good correlation has been observed between the permeation coefficient and the partition coefficient, log P (r=0.951). In the second part, albendazole-loaded nanoparticles (about 300 nm) prepared by the emulsion solvent evaporation method have shown a sufficient entrapment efficiency (36.4 +/- 6.4%) to raise the apparent solubility of albendazole. The diffusion of drug from the nanoparticles across the hydatid cyst membrane was also improved compare to albendazole suspension. These results have shown the interest of the albendazole-loaded nanoparticles for the treatment of hydatid cysts in the future.