Intracellular delivery mediated by an ethosomal carrier.

The goal of this work was to investigate the efficiency of transcellular delivery into Swiss albino mice 3T3 fibroblasts of molecules with various physico-chemical characteristics from ethosomes, phospholipid vesicular carriers containing ethanol. The probes chosen were: 4-(4-diethylamino) styryl-N-methylpyridinium iodide (D-289), rhodamine red dihexadecanoylglycerophosphoethanolamine (RR) and fluorescent phosphatidylcholine (PC*). The penetration of these fluorescent probes into fibroblasts and nude mice skin was examined by CLSM and FACS. CLSM micrographs showed that ethosomes facilitated the penetration of all probes into the cells, as evident from the high-intensity fluorescence. In comparison, when incorporated in hydroethanolic solution or classic liposomes, almost no fluorescence was detected. The intracellular presence of each of the three probes tested, was evident after 3 min of incubation. Furthermore, with ethosomal D-289, fluorescence was also seen in the fibroblast nucleus. Enhanced delivery of molecules from the ethosomal carrier was also observed in permeation experiments with the hydrophilic calcein and lypophilic RR to whole nude mouse skin. Calcein penetrated the skin to a depth of 160, 80 and 60 microm from ethosomes, hydroethanolic solution and liposomes, respectively. Maximum fluorescence intensities measured for RR delivered from ethosomes, hydroethanolic solution and liposomes were 150, 40 and 20 AU, respectively. Fibroblast viability tests showed that the ethosomal carrier is not toxic to the cultured cells.

Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier.

The main objective of the present work was to investigate the dermal and intracellular delivery of bacitracin, a model polypeptide antibiotic, from ethosomes. Bacitracin and fluorescently labeled bacitracin (FITC-Bac) ethosomes were characterized for shape, lamellarity, fluidity, size distribution and entrapment capacity by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and ultracentrifugation, respectively. Confocal laser scanning microscopy (CLSM) experiments revealed that ethosomes facilitated the copenetration of antibiotic and phospholipid into cultured 3T3 Swiss albino mice fibroblasts. These results, confirmed by data obtained in fluorescent-activated cell sorting (FACS) experiments, suggest that ethosomes penetrate cellular membrane releasing the entrapped molecule within cells. Additional work was focused on skin permeation behavior of FITC-Bac from ethosomal systems in in vitro and in vivo experiments through human cadaver and rat skin, respectively. These studies demonstrated that the antibiotic peptide was delivered into deep skin layers through intercorneocyte lipid domain of stratum corneum (SC). Occlusion had no effect on the permeation profile of the drug from ethosomes in in vitro experiments. Efficient delivery of antibiotics to deep skin strata from ethosomal applications could be highly beneficial, reducing possible side effects and other drawbacks associated with systemic treatment. Furthermore, ethosomal delivery systems could be considered for the treatment of a number of dermal infections, requiring intracellular delivery of antibiotics, whereby the drug must bypass two barriers: the SC and the cell membrane.

Oleic acid, a skin penetration enhancer, affects Langerhans cells and corneocytes.

Permeation enhancers (PE) are frequently used in the field of dermal research and for the development of transdermal delivery products. However, their influence on skin epidermal Langerhans cells (LC) has not yet been investigated. In this work we studied the effect of four PE, oleic acid (OA), propylene glycol (PG), ethanol, and diethylene glycol monoethyl ether (DGME), and an iontophoretic treatment on the morphometric parameters of epidermal Langerhans cells (LC). Retinoic acid (RA) was used as a positive control. Test solutions were applied to the footpad of Sabra mice. The area, perimeter, density and shape factor (SF) were the morphometric parameters evaluated following ATPase staining of LC. Application of RA led to a large decrease in cell density (-50.2%, P<0.01) and dendritic shape (19.8%, P<0.01). Treatment with 10% OA in ethanolic solution caused a severe decrease in LC density (-69.0%, P<0.01), accompanied by a decrease in dendricity as measured by the changes in SF. Ethanol had no statistically significant effect on the LC morphologic parameters tested. All other PE had a mild, if any, effect on LC morphology. SEM micrographs of the skin of IOPS hairless rats demonstrated that 24 h in vivo treatment with 10% OA in ethanolic solution resulted in the generation of pores on the surface of epidermal corneocytes.

Ethosomes - novel vesicular carriers for enhanced delivery: characterization and skin penetration properties.

This work describes a novel carrier for enhanced skin delivery, the ethosomal system, which is composed of phospholipid, ethanol and water. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth, than either liposomes or hydroalcoholic solution. The ethosomal system dramatically enhanced the skin permeation of minoxidil in vitro compared with either ethanolic or hydroethanolic solution or phospholipid ethanolic micellar solution of minoxidil. In addition, the transdermal delivery of testosterone from an ethosomal patch was greater both in vitro and in vivo than from commercially available patches. Skin permeation of ethosomal components, ethanol and phospholipid, was demonstrated in diffusion-cell experiments. Ethosomal systems composed of soy phosphatidylcholine 2%, ethanol 30% and water were shown by electron microscopy to contain multilamellar vesicles. 31P-NMR studies confirmed the bilayer configuration of the lipids. Calorimetry and fluorescence measurements suggested that the vesicular bilayers are flexible, having a relatively low T(m) and fluorescence anisotropy compared with liposomes obtained in the absence of ethanol. Dynamic light scattering measurements indicated that ethanol imparted a negative charge to the vesicles. The average vesicle size, as measured by dynamic light scattering, was modulated by altering the ethosome composition. Experiments using fluorescent probes and ultracentrifugation showed that the ethosomes had a high entrapment capacity for molecules of various lyophilicities.

Cannabidiol-transdermal delivery and anti-inflammatory effect in a murine model.

Cannabidiol (CBD) is a new drug candidate for treatment of rheumatic diseases. However, its oral administration is associated with a number of drawbacks. The objective of this study was to design a transdermal delivery system for CBD by using ethosomal carriers. CBD ethosomes were characterized by transmission electron microscopy, confocal laser scanning microscopy and differential scanning calorimetry. Results indicated that CBD and phosphatidylcholine form an eutectic mixture. In vivo application of ethosomal CBD to CDI nude mice produced a significant accumulation of the drug in the skin and in the underlying muscle. Upon transdermal application of the ethosomal system to the abdomen of ICR mice for 72 h, steady-state levels were reached at about 24 h and lasted at least until the end of the experiment, at 72 h. Furthermore, transdermal application of ethosomal CBD prevented the inflammation and edema induced by sub-plantar injection of carrageenan in the same animal model. In conclusion, ethosomes enable CBD's skin permeation and its accumulation in a depot at levels that demonstrate the potential of transdermal CBD to be used as an anti-inflammatory treatment.

Multiplication végétative in vitro de Gardenia jasminoïdes Ellis: In vitro Vegetative Multiplication of Gardenia jasminoïdes Ellis.

The vegetative propagation in vitro of Gardenia jasminoides Ellis, an ornamental shrub producing secondary metabolites useful in perfumery and pharmacy, has been investigated. In the presence of benzylaminopurine (0.3 to 1 mg l(-1)) and indoleacetic acid (1 mg l(-1)), axillary shoots arose from nodes of greenhause plants, but a decrease of morphogenic activity was noticed in subcultures. Mother plants have been developed in vitro from which shoots may be taken with the most effective system being a monthly subculture of nodal explants. As many as one million shoots per year could, theoretically, be produced from one shoot grown in vitro. Rooting occurred easily in three weeks in vitro in the presence of indoleacetic acid (1 mg l(-1)) and charcoal (2 g l(-1)) or in vivo by soaking the base of the shoot in the same auxin for two hours. After acclimatization, the rooted plants developed and flowered normally. The successive stages have been sufficiently well monitored to be used as an effective means of vegetative propagation.

Active curcumin nanoparticles formed from a volatile microemulsion template.

We report on biological performance of organic nanoparticles formed by a simple method based on rapid solvent removal from a volatile microemulsion. The particular focus of the study was on testing the suitability of the method for substances soluble in partially water-miscible organic solvents as well as on evaluating the therapeutic activity of the resultant nanoparticles. Curcumin was employed as a model for hydrophobic drug, and, as it is soluble in water-miscible organic solvents, it was successfully incorporated into a new cyclopentanone-water microemulsion system. During rapid solvent removal by spray-drying, the nanometric droplets of the microemulsion were converted into nanoparticles containing amorphous curcumin with the average size of 20.2±3.4 nm, having ζ potential of -36.2 ±1.8 mV. These nanoparticles were dispersible in water and retained the high loading of the active substance. The therapeutic activity of the resulting nanoparticles was demonstrated in a pancreatic cancer cell line Panc-1. The effective concentration for reducing the metabolic activity was found to be 11.5 µM for nanoparticles compared with 19.5 µM for free curcumin.

Size and shape effects in the biodistribution of intravascularly injected particles.

Understanding how size and shape can affect the biodistribution of intravascularly injected particles is of fundamental importance both for the rational design of delivery systems and from a standardization and regulatory view point. In this work, uncoated silica spherical beads, with a diameter ranging from 700 nm to 3 microm, and uncoated non-spherical silicon-based particles, with quasi-hemispherical, cylindrical and discoidal shapes, have been injected into tumor bearing mice. The number of particles accumulating in the major organs and within the tumor mass has been measured through elemental silicon (Si) analysis. For the spherical beads, it has been found that the number of particles accumulating in the non-RES organs reduces monotonically as the diameter d increases, suggesting the use of smaller particles to provide a more uniform tissue distribution. However, discoidal particles have been observed to accumulate more than others in most of the organs but the liver, where cylindrical particles are deposited at a larger extent. These preliminary results support the notion of using sub-micrometer discoidal particles as intravascular carriers to maximize accumulation in the target organ whilst reducing sequestration by the liver.

In vivo evaluation of safety of nanoporous silicon carriers following single and multiple dose intravenous administrations in mice.

Porous silicon (pSi) is being extensively studied as an emerging material for use in biomedical applications, including drug delivery, based on the biodegradability and versatile chemical and biophysical properties. We have recently introduced multistage nanoporous silicon microparticles (S1MP) designed as a cargo for nanocarrier drug delivery to enable the loaded therapeutics and diagnostics to sequentially overcome the biological barriers in order to reach their target. In this first report on biocompatibility of intravenously administered pSi structures, we examined the tolerability of negatively (-32.5±3.1mV) and positively (8.7±2.5mV) charged S1MP in acute single dose (10(7), 10(8), 5×10(8) S1MP/animal) and subchronic multiple dose (10(8) S1MP/animal/week for 4 weeks) administration schedules. Our data demonstrate that S1MP did not change plasma levels of renal (BUN and creatinine) and hepatic (LDH) biomarkers as well as 23 plasma cytokines. LDH plasma levels of 145.2±23.6, 115.4±29.1 vs. 127.0±10.4; and 155.8±38.4, 135.5±52.3 vs. 178.4±74.6 were detected in mice treated with 10(8) negatively charged S1MP, 10(8) positively charged S1MP vs. saline control in single and multiple dose schedules, respectively. The S1MPs did not alter LDH levels in liver and spleen, nor lead to infiltration of leukocytes into the liver, spleen, kidney, lung, brain, heart, and thyroid. Collectively, these data provide evidence of a safe intravenous administration of S1MPs as a drug delivery carrier.

A new approach for treatment of deep skin infections by an ethosomal antibiotic preparation: an in vivo study.

OBJECTIVES: Dermal and subdermal bacterial infections, caused mainly by Staphylococcus aureus, are currently treated by systemic antibiotics. The aim of the present study was to investigate a new approach to treat deep skin and soft tissue bacterial infections by dermal application of erythromycin in an ethosomal carrier. METHODS: A model for deep dermal S. aureus infection in mice was developed. The efficiency of ethosomal erythromycin applied to the skin-infected site was compared with intraperitoneal erythromycin administration and with local application of hydroethanolic erythromycin solution. The parameters evaluated were the development of dermal wound, histological sections and bacterial count of the infected tissue. RESULTS: The in vivo experiments demonstrated a very efficient healing of S. aureus-induced deep dermal infections when the mice were treated with ethosomal erythromycin. Bacterial counts and histological evaluation of the skin treated with ethosomal antibiotic revealed no bacterial growth and normal skin structure. On the contrary, no subdermal healing was observed in infected animals treated with topical hydroethanolic erythromycin solution. In this group, animals developed deep dermal abscesses and the dermal structures were destroyed where S. aureus colonies were present. Bacterial counts of the infected tissues were 1.06 x 10(7) and 0.27 x 10(7) cfu/g of tissue, respectively, on days 7 and 10. CONCLUSIONS: Therapy with ethosomal erythromycin applied to the skin of S. aureus-infected mice was as effective as systemically administered erythromycin, suggesting a new possibility to treat deep dermal infections by local application of antibiotic in ethosomal carrier.

Construction and use of a replication-competent human immunodeficiency virus (HIV-1) that expresses the chloramphenicol acetyltransferase enzyme.

The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.

Structure of two maize phytase genes and their spatio-temporal expression during seedling development.

Up to 80% of Zea mays L. grain phosphorus is stored in the form of phytin in the embryo. Our objective is to determine the control of phytin mobilization during germination and seedling growth. A maize phytase cDNA, phy S11, has been previously characterized (Maugenest et al., Biochem J 322: 511-517, 1997). In the present work, phy S11 was used to screen a maize genomic library and two distinct genes, PHYT I and PHYT II, were isolated and sequenced. The transcribed sequences of these two genes presented a strong homology whereas the untranscribed upstream and downstream sequences appeared very different. Northern blot analysis and in situ hybridization showed a high accumulation of phytase mRNA at the early steps of germination in the coleorhiza, radicle cortex and coleoptile parenchyma. Phytase expression was also detected at a lower extent in the scutellum. In adult plants, northern blot analyses revealed low but significant levels of phytase mRNA in the roots. In situ hybridizations on root cross-sections localized phytase mRNA in rhizodermis, endodermis and pericycle layers. Immunolocalization analysis showed phytase accumulation at the same sites as its mRNA. A RT-PCR approach was used in an attempt to discriminate between the transcripts from each gene in the different situations. These experiments indicate that both genes are expressed during germination, whereas only PHYT I is expressed in adult roots. This suggests that signals responsible for phytase gene expression in roots are different from those responsible for gene expression during germination.