Interaction of Liposomes with the Human Skin Lipid Barrier: hSclls as Model System — DSC of Intact Stratum Corneum and in Situ CLSM of Human Skin

Abstract

The interaction of liposomes with the human skin lipid barrier was studied by (i) physico-chemical analyses of lipid vesicles prepared from the complete mixture of human epidermal lipids, (ii) differential scanningcalorimetry of integral horny layers (strata cornea) before and after topical administration of phospholipid vesicles and (iii) confocal laser scanning microscopy of pieces of skin to which Rho-PE labelled DMPC-liposomes were applied. The data imply that the epidermal lipids of the human skin have at body temperature a high propensity of mixing if Ca²⁺ -ions are present, that intact liposomes do not penetrate into the viable epidermis but diffusing/mixing liposomal lipids disorder the complex structure of the upper intercellular lipid sheets thereby causing an increased hydration. The result is an enhancement of penetration of chemicals via the ‘polar and lipid routes'.     

Controlled Topical Delivery of Cyclosporin-a from Nonionic Liposomal Formulations: Mechanistic Aspects

Abstract

In a previous report (1), we showed that the rate and extent of uptake of cyclosporin-A (CsA) following topical application of nonionic liposomal formulations composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10) into and through hairless mouse skin mounted on Franz diffusion cells could be controlled by varying the ratios of GDL to POE-10 (CH being held constant at 15 wt%). However, the pathways of transport as well as the dominant factors that control drug delivery from these formulations are not well understood. In this report, we describe results from studies similar in design to that reported earlier but using the melted form of the lipid components as a vehicle for transport of CsA into and through hairless mouse skin. The results suggest that the transport of CsA from liposomal formulations into and through the skin occurs as a result of dehydration of the liposomes followed by melting of the lipid components on the skin. Microautoradiographic studies suggest that CsA is predominantly transported via the pilosebaceous pathway.     

Enhanced skin targeting of retinoic acid spanlastics: in vitro characterization and clinical evaluation in acne patients

Abstract

Acne vulgaris is the most common dermatological disorder affecting millions of individuals. Acne therapeutic solutions include topical treatment with retinoic acid (RA) which showed a good efficacy in treatment of mild and moderate cases. However, the high prevalence of adverse events, such as skin dryness, shedding and skin irritation affects the patient convenience and obstruct the acne treatment. Thus, the objective of this paper was to produce Span 60 based elastic vesicles enriched with penetration enhancers, and study their influence on the delivery of RA and its skin irritation. RA-loaded nanovesicles, enriched with Transcutol^®/Labrasol^®, were made using the thin film hydration technique, and assessed for entrapment efficiency, particle size and zeta potential. The optimized RA-loaded nanovesicles (composed of Span 60-Tween 20, and Transcutol^®) were morphologically assessed via transmission electron microscopy. Moreover, RA deposition into newborn mice skin was assessed in vitro under non-occlusive conditions, where the optimized RA-loaded nanovesicles showed 2-fold higher RA deposition in the skin compared to the corresponding one lacking Transcutol. The optimized RA-loaded nanovesicles incorporated into 1% carbopol gel was evaluated for in-vivo clinical performance in acne patients, and showed appreciable advantages over the marketed formulation (Acretin^®) in the treatment of acne regarding skin tolerability and patient’s compliance.     

Sex and a snail's sperm: on the transport,storage and fate of dimorphic sperm in Littorinidae

Summary

Littorinid parasperm develop from a distinctive lineage of germ cells which exhibit a process of nuclear destruction that has apototic characteristics. Fragments of DNA and other nuclear breakdown products are incorporated into secretion granules in parasperm that ultimately find their way to the female bursa copulatrix. Spermatozeugmata are stored in the seminal vesicles and, if unused during the breeding season, they are recycled by phagocytosis. Attachment between eusperm and parasperm is facilitated by an electrostatic interaction of proteins. Detachment, caused by alkaline prostate fluid, occurs by the time the ejaculate reaches the tip of the penis. Thus transport of eusperm by parasperm to the female is unlikely. parasperm are sterile cells that may function in defense against rival eusperm as suggested by the presence of lysosomes, or they may act as nuptial gifts as they are packed with glycoprotein nutrients. Differences in the reactivity of different parasperm to specific lectins may enable separation of dimorphic sperm by lectin affinity chromatography, thereby facilitating future studies on individual parasperm. In female Littorinidae, sperm are stored incapacitated in storage organs, or rarely in the ovary itself. In L. littorea serotonin caused spawning of unencapsulated eggs, which, in the presence of activated sperm, became polyspermic.     

Cytinus hypocistis L. embryogenesis: Some biological and ultrastructural aspects of fertilization and embryo development

Abstract

The present paper examines some biological and ultrastructural aspects of fertilization and early development of the embryo in Cytinus hypocistis, a parasitic plant belonging to the Rafflesiaceae. The probable functions of a mucilaginous substance contained in the ovary and embedding the numerous pollen tubes coming from the style are discussed.

It was ascertained that pollen tubes pass through the micropyle and enter a synergid pushing, their way through the nucellar cells that show swollen walls owing to a probable enzymatic action whose function is to facilitate pollen tube penetration. It was hypothesized that the secretion of such enzymes is attributable to the numerous pollen present in the ovary or entering the mycropyle.

Since, in all the ovules observed, synergid degeneration was never found before the arrival of the pollen tube, this degeneration was interpreted as being caused by the material disharged by the pollen tube, rather than being an essential prerequisite for pollen tube penetration into the synergid.

Pollen tube content was observed to be made up of an intensely electron-dense substance surrounding many lipidic globules and numerous polysaccharide vesicles that fuse with the pollen tube wall, clearly contributing to its growth.

The sequence of the first divisions of the developing embryo was followed and the extreme reduction of the embryo is confirmed.

In Cytinus hypocistis starch is totally absent from all the sells belonging to the female gamethophyte as well as to those belonging to the embryo, but lipidic globules are very frequent; it is therefore supposed that these bodies constitute good material for the nutrition of the zygote and early embryo.     

Predicting doxorubicin drug delivery by single-walled carbon nanotube through cell membrane in the absence and presence of nicotine molecules: a molecular dynamics simulation study

Abstract

In order to study the interaction of the anticancer agent Doxorubicin with the single-walled carbon nanotubes with different diameters as drug delivery systems, the molecular dynamics (MD) simulations have been used. Also, for design and development of intracellular Doxorubicin drug delivery systems, a series of steered MD simulations are applied to explore the possibility of encapsulated Doxorubicin–carbon nanotube penetration through a lipid bilayer in presence and absence of Nicotine molecules at different pulling rates. Our simulation results showed that in spite of the adsorption of drug molecules on the outer sidewall of the nanotubes, the spontaneous localization of one Doxorubicin molecule into the cavity of the nanovectors with larger diameters is observed. It is found that the presence of Nicotine molecules in extracellular medium increases the required force for pulling nanotube-encapsulated drug as well as the required time for penetration process, especially at higher velocity. Also, the entering process of the Nicotine molecules into the carbon nanotube causes that the encapsulated drug molecule is fully released in the hydrophobic phase of the lipid bilayer.

Communicated by Ramaswamy H. Sarma     

Investigating the penetrating potential of nanocomposite β-cycloethosomes: development using central composite design,in vitro and ex vivo characterization

Context: Atopic dermatitis (AD) is a chronic skin disease characterized by inflammation of the skin and has exhibited remarkable repercussions on human life across the globe. Fluocinolone acetonide (FA), a topical corticosteroid is employed in the treatment of atopic dermatitis, but suffers from limited penetration into deeper epidermis of atopic skin.

Objective: The present investigation was focused to explore the utility of β-cylcoethosomes in improvising the penetration deep into the skin.

Materials and methods: β-Cylcoethosomes developed using β-cycloamylose by injection method were evaluated for vesicle size, entrapment efficiency and in vitro release. Central Composite design employed for the preparation depicted FA8 as an optimized formulation which was then formulated as dermatological gel using carbomer 934P as a gel base. The gels were characterized for pH, viscosity, drug content and in vitro permeability.

Results and discussion: Optimized formulation (FA8) showed maximum desirability (0.795) with vesicle size of 228.33?±?1.23?nm), EE (82.49?±?1.21%) and CDR (90.90?±?0.29%). FA8-loaded gels showed maximum in vitro permeability as found in BG and BGP (83.22?±?0.72% and 84.02?±?0.87). BG was selected as an optimized gel and compared with optimized reference ethosomal gel and control gel. CLSM studies depicted deeper uniform penetration of fluorescent dye deep into the epidermis via BG. Improved penetration was observed due to the synergistic effect exerted by ethanol and β-cycloamylose.

Conclusion: β-cylcoethosomes proved to be a promising carrier for improvised penetration of fluocinolone acetonide via topical gel.     

Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis

The present study aimed to evaluate the potential of liposomes loaded with paromomycin (PA), an aminoglycoside antibiotic associated with poor skin penetration, for the topical treatment of cutaneous leishmaniasis (CL). Fluid liposomes were prepared and characterized for particle size, zeta potential, and drug entrapment. Permeation studies were performed with two in vitro models: intact and stripped skin. The antileishmanial activity of free and liposomal PA was evaluated in BALB/c mice infected by Leishmania (L.) major. Drug entrapment ranged from 10 to 14%, and the type of vesicle had little influence on this parameter. Particle size and polydispersity index of the vesicles composed by phosphatidylcholine (PC) and PC/cholesterol (Chol) ranged from of 516 to 362?nm and 0.7 to 0.4, respectively. PA permeation across intact skin was low, regardless of the formulation tested, while drug penetration into skin (percent of the applied dose) from PC (7.2?±?0.2%) and PC/Chol (4.8?±?0.2%) liposomes was higher than solution (1.9?±?0.1%). PA-loaded liposomes enhanced in vitro drug permeation across stripped skin and improved the in vivo antileishmanial activity in experimentally infected mice. Our findings suggest that the liposomes represent a promising alternative for the topical treatment of CL using PA.     

Seed–seedling conflict in conifers as a result of plant–plant interactions at the forest-tundra ecotone

Background: As the climate warms, plant interactions between shrubs and conifer seedlings may affect migration of boreal trees into alpine areas; however these interactions have not been widely tested across conifer life stages.

Aims: Determine the role of shading by Betula glandulosa shrubs on early Picea mariana recruitment in the forest-tundra ecotone of the Mealy Mountains, Labrador (Canada).

Methods: Four shrub treatments were established with varying degrees of shade (unaltered, trimmed, cleared, shaded). Emergence and growth of P. mariana in treatment plots were compared to control plots without shrubs.

Results: Seedling emergence was facilitated; seedlings beneath shrubs emerged at greater levels (16% ± 5.1%) than in the control (0.5% ± 0.3%); however, evidence of facilitation post-emergence was weak, likely due to stronger interactions with seedbed species at this stage. Vertical growth of young seedlings was greatest in control and shaded plots, but needle production significantly decreased with increased shade suggesting that seedlings may compete with shrubs for light early in life.

Conclusions: Although facilitation has been previously implicated as an important mechanism in stressful environments, our study indicates that the early life stages of some conifers at the tree line are not strongly facilitated by shrubs as has been previously suggested for adults.     

Aggregation and Phospholipid Intermixing of Oleic-Acid-Containing Phosphatidylcholine Vesicles Induced by Polylysine

Abstract

Polylysine induced aggregation and phospholipid intermixing between small unilamellar vesicles of egg yolk phosphatidylcholine containing free oleic acid. the process was dependent on pH, being attributed to the presence of oleic acid. Neither intermixing nor leakage of the encapsulated aqueous contents was detected, nor did the size of such vesicles increase after treatment with polylysine. the maximum value of phospholipid intermixing was about 50%. these results are interpreted as representing reversible hemifusion between vesicles, without total membrane fusion.     

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

Abstract

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe₃O₄), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe₃O₄ nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60?°C, free lipase lost its initial activity within 80?min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120?min of heat treatment at 60?°C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).     

Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes

Summary After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4-diisothiocyano-2,2-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976,Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.This paper is dedicated to the memory of Walther Wilbrandt.     

Effect of Lipid Dose on the Redistribution and Blood Pool Clearance Kinetics of Peg-Modified Technetium-Labeled Lipid Vesicles

Abstract

The current literature reports that the circulation half-life of PEG-modified vesicles is independent of lipid dose over the range of β4-400 μmol/ kg. The results presented in this paper indicate that PEG-modified vesicles exhibit a dose-dependent circulation half-life at even lower lipid doses. At lipid doses of 2.13, 1.30, 0.50 and 0.16 μmol/kg, the half-life for the blood pool clearance of activity of technetium-99m encapsulated in PEG-coated vesicles was approximately 20, 16, 4, and 2 hr respectively. There was no statistically significant difference in clearance half-life for lipid doses of 2.13 and 1.30 μmol/kg (Kruskal-Wallis Anova, p>0.05). These results suggest that the dose-independent range may be extended down to approximately 1 (μmol/kg but that at lower doses, the circulation half-life is dependent on the lipid dose. These results are discussed in terms of the mechanism of clearance of activity from the circulation and the potential utility of PEG-modified technetium-99m labeled vesicles as blood pool imaging agents for nuclear medicine.     

Clathrin: Its Role in Receptor-Mediated Vesicular Transport and Specialized Functions in Neurons

Abstract

Clathrin constitutes the coat of vesicles involved in three receptor-mediated intracellular transport pathways; the export of aggregated material from the trans-Golgi network for regulated secretion, the transfer of lysosomal hydrolases from the trans-Golgi network to lysosomes and receptor-mediated endocytosis at the plasma membrane. The clathrin subunits and the other major coat constituents, the adaptor polypeptides, interact in specific ways to build the characteristic polygonal clathrin lattice and to attach the coat to integral membrane receptors. Both clathrin coat assembly and disassembly on the cytoplasmic side of the membrane are multistep processes that are regulated by the coat constituents themselves and by cytosolic proteins and factors. Neurons represent a cell type with distinct morphology and special demands on exocytic and endocytic pathways that requires neuron-specific constituents and modifications of clathrin-coated vesicles.     

Growth measurement of surface colonies of bacteria using augmented reality

ABSTRACT

The provision of hands-on wet lab training for students to perform micro-organism proliferation experiments is useful for experiential skills development. However, the risk of laboratory-acquired infections arising from inadvertent ingestion, inhalation or skin penetration of these micro-organisms presents a safety concern. To obviate this risk, an augmented reality exercise requiring only a tablet/smartphone, and a petri dish with appropriate markers, was devised to allow students to study Escherichia coli colony growth on agar plates. Positive responses were obtained from a pilot study conducted with high school and undergraduate students on its use.     

Ultradeformable liposomes with terpenes for delivery of hydrophilic compound

Ultradeformable liposomes containing penetration enhancers were created to deliver NaFl. Vesicles were investigated for their particle size, zeta potential, NaFl entrapment efficiency (%EE), loading efficiency, and in vitro skin penetration. The vesicles obtained were spherical in shape, with a particle size of less than 100 nm and a negative surface charge (–6 to –11 mV). The %EE of NaFl loaded in vesicles ranged from 37 to 48%. Ultradeformable liposomes with monoterpenes (d-limonene, 1,8-cineole and geraniol) significantly improved NaFl penetration through the skin. Confocal laser scanning microscopy analysis confirmed skin-penetration results and was used to evaluate the behavior of hydrophilic compounds penetrating through the skin.     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Precision parameters from spin-probe studies of membranes using a partitioning technique. Application to two model membrane vesicles

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.