Jui Chen Tsai

Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in mouse embryonic fibroblasts

With the advancement of nanotechnology, nanomaterials have been comprehensively applied in our modern society. However, the hazardous impacts of nanoscale particles on organisms have not yet been thoroughly clarified. Currently, there exist numerous approaches to perform toxicity tests, but common and reasonable bio-indicators for toxicity evaluations are lacking. In this study, we investigated the effects of silver nanoparticles (AgNPs) on NIH 3T3 cells to explore the potential application of these nanoparticles in consumer products. Our results demonstrated that AgNPs were taken up by NIH 3T3 cells and localized within the intracellular endosomal compartments. Exposure to AgNPs is a potential source of oxidative stress, which leads to the induction of reactive oxygen species (ROS), the up-regulation of Heme oxygenase 1 (HO-1) expression, apoptosis and autophagy. Interestingly, AgNPs induced morphological and biochemical markers of autophagy in NIH 3T3 cells and induced autophagosome formation, as evidenced by transmission electron microscopic analysis, the formation of microtubule-associated protein-1 light chain-3 (LC3) puncta and the expression of LC3-II protein. Thus, autophagy activation may be a key player in the cellular response against nano-toxicity.

Permeability barrier disruption alters the localization and expression of TNFα/protein in the epidermis

Previous studies have shown that (1) epidermal TNFα mRNA levels are increased following acute disruption of the cutaneous permeability barrier; (2) this increase is maximal at 1 h and decreases to control levels by 8 h; and (3) in essential fatty acid-deficient (EFAD) mice, a chronic model of barrier perturbation, TNFα mRNA levels are also elevated several-fold over controls. In the present study we determined, using immunocytochemical procedures, epidermal TNFα protein levels following either acute of chronic barrier disruption and the localization of any increase. Frozen, paraffin and Antibed sections of skin were incubated with polyclonal anti-mouse TNFα antisera and detection was accomplished by either immunoperoxidase or fluorescence procedures. We found that (1) TNFα-immunoreactive protein was present in normal mouse epidermis, and was primarily localized to the upper nucleated layers where it displayed a diffuse cytosolic pattern; (2) acute disruption of the barrier with acetone or tape-stripping resulted in TNFα staining that was more intense throughout all of the nucleated epidermal cell layers in comparison with normal epidermis; (3) the increase in TNFα staining occurred as early as 2 h after barrier disruption; and (4) increased TNFα staining was also observed in the stratum corneum of EFAD mice. These results indicate that epidermal TNFα protein levels increase after both acute and chronic barrier disruption, and are consistent with the hypothesis that TNFα may signal and/or coordinate portions of the cutaneous response to barrier disruption.

Human skin surface lipid film: An ultrastructural study and interaction with corneocytes and intercellular lipid lamellae of the stratum corneum

Sebum is a complex mixture of lipids, which is secreted by mammalian sebaceous glands, and forms a fluid film over the skin surface. After sebum is secreted, it becomes mixed with lipid from the keratinizing epithelium and forms the skin surface lipid film (SSLF). Until now, direct fine structural observation of the SSLF has been lacking. In the present work, we viewed the detailed structures of the human SSLF by ruthenium tetroxide staining. The results showed that the SSLF formed an amorphous sheet of variable thickness on the skin surface instead of forming lipid droplets, as had been the usual assumption. In general, its thickness was < 0.5 μm or even negligible in sebum‐poor extremities. However, in the sebum‐rich face, its thickness was > 4 μm in focal areas. Consistent with the thickness of SSLF, the sebum quantity showed great regional variation. It varied from 1 μg/cm2 (leg) to 189 ± 42.7 μg/cm2 (mean ± SD: face). The SSLF was composed of numerous fine granules of about 4–5 nm in a random orientation. Within the SSLF, variable amounts of deranged lipid lamellae derived from corneocytes were mixed with sebum. As well as on the skin surface, a similar amount of sebum was also found between the desquamating corneocytes in the uppermost several layers of the stratum corneum (SC). We also observed the presence of intercellular lipid lamellae in the outer layers of the SC: their lipid envelope remained intact even in desquamated corneocytes. Our results provide some new insights concerning the structure of the SSLF and its relationship with the SC.

Distribution of salicylic acid in human stratum corneum following topical application in vivo: a comparison of six different formulations

Distribution of salicylic acid in human stratum corneum from treatment of six different formulations was assessed by quantitation of drug content in sequentially tape-stripped stratum corneum after a single 2-h dose was applied unoccluded to skin on the ventral forearm of four female subjects. The profile and total amounts of stratum corneum removed in 20 tape-strips varied among different types of formulations. With or without normalization by the total stratum corneum weights removed, the extent of drug delivery to the stratum corneum decreased in the following order: SA (5%) > > SAC (10%), Duofilm (16.7%) > TSSS (2%) > SAO (10%), Salic (2.5%), the percentage in parentheses indicating the salicylic acid concentration in each formulation. The greatest topical bioavailability was observed for the alcoholic solution containing glycerol (SA). The 10% collodion formulation (SAC) was found to deliver an amount of salicylic acid into the stratum corneum 2-fold greater than 10% ointment formulation (SAO). Use of absorption ointment (TSSS) also increased the uptake of salicylic acid into the stratum corneum in comparison with formulations based on simple ointment (SAO) and oil in water (o/w) cream (Salic). The partitioning of salicylic acid from collodion formulations (SAC and Duofilm) appeared to be concentration-independent. The results of this study indicate that topical bioavailability of salicylic acid in the stratum corneum varies substantially among different formulations.

Effect of minoxidil concentration on the deposition of drug and vehicle into the skin

Summary Minoxidil dissolved in propylene glycol/ethanol/water (20: 60: 20; v/v) was more efficiently delivered into hairless mouse skin from a 0.02% solution than from a 2% formulation during in vitro mass balance experiments. Evaporation of the vehicle after application of the 2% minoxidil solution led to compositional changes so drastic that saturation is attained quickly. The shifting compositions of the vehicle were experimentally profiled and the drug’s solubility was measured in media reflecting these changes over time. The original 2% formulation has excess capacity to dissolve minoxidil but, following its application, the solution rapidly saturates through evaporation, reaching saturation within 30 minutes. While the 2% formulation definitely functions at a proportionally higher thermodynamic activity than the 0.02% formulation at early times, most of this thermodynamic advantage is lost abruptly upon precipitation of the drug. The driving force following drug precipitation only reflects the dissolved minoxidil, and thus the relative delivery rate of the 2% formulation eventually drops to only several multiples of the delivery rate of the 0.02% solution which never quite attains saturation.

Nasopharyngeal carcinoma-susceptibility locus is localized to a 132 kb segment containing HLA-A using high-resolution microsatellite mapping.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor uniquely prevalent in southern Chinese. HLA‐A2 is associated with NPC. In a previous study, we showed that the genes associated with susceptibility to NPC are primarily located within the HLA‐A locus in Taiwanese NPC patients. However, the pathogenic genes causing NPC susceptibility remain unknown. Here, 8 polymorphic microsatellite markers distributed over a 1 megabase region surrounding the HLA‐A locus were subjected to genetic analysis for the NPC‐susceptibility locus. Statistical studies of associated alleles detected on each microsatellite locus showed that the NPC‐ susceptibility genes are most likely located between the D6S510 and D6S211 markers within a 132 kb segment containing the HLA‐A locus. These results undoubtedly would facilitate the further positional cloning of the NPC‐susceptibility locus, which has been elusive for the past 30 years.

Influence of Application Time and Formulation Reapplication on the Delivery of Minoxidil through Hairless Mouse Skin as Measured in Franz Diffusion Cells

Relationships are drawn between the extent of topical delivery of test compounds in solution and the period of residence of their formulation on the skin. The studies were performed using in vitro diffusion cell techniques and a test formulation containing 2% 3H-minoxidil dissolved in 60% ethanol, 20% water and 20% 14C-propylene glycol. The permeation of propylene glycol was effectively halted upon cleansing the skin surface; the skin had very little reservoir capacity for this substance. However, the rate of delivery of minoxidil was only slowed but not stopped upon cleansing. The suggestion here is that a reservoir of minoxidil is formed in the skin which is capable of sustaining an appreciable input of drug even after the skins surface is scrupulously cleaned. Assay of epidermal concentrations of these species not only confirms the existence of the minoxidil reservoir but also shows that the degree of its tissue concentration is proportional to the time of residence of the formulation on the skin surface. Reapplication of blank vehicle to the cleansed surface had little to no effect on the permeation of the minoxidil and was similarly without effect on that of propylene glycol. While it comes as no surprise that formulation residence time is an important variable in topical delivery, this study demonstrates the complexities of quantitative dependencies of delivery on residence time.

Noninvasive characterization of regional variation in drug transport into human stratum corneum in vivo

AbstractPurpose. To investigate the mechanisms underlying the regional variations in drug transport into human stratum corneum (SC) of two model compounds of different lipophilicity and molecular size, 4-cyanophenol (CP) and cimetidine (CM), in vivo by non-invasive, quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Methods. Saturated solutions of CP and CM were applied to the skin surface of eleven Chinese men, at five anatomical sites, including forearm, back, thigh, leg, and abdomen, for 10-15 min and 3-5 h, respectively. After the skin surface was cleansed of remaining chemicals, the SC was tape-stripped sequentially up to 20 times, and the drug concentration profiles in the tape-stripped SC were determined using ATR-FTIR spectroscopy. Thickness of the SC was estimated simultaneously using two-point measurements of transepidermal water loss before and after completion of tape stripping. Estimation of partition, diffusion, and permeability coefficients was achieved by analysis of the data using the unsteady-state diffusion equation. Results. The rank orders of regional variation in partition and diffusion coefficients of CP and CM were different. The rank order of regional variation in permeability coefficients was similar for both drugs and decreased in the order of back > forearm > thigh > leg ≥ abdomen, but the variation was more prominent for CM. Conclusions. Regional variation in SC transport of CP was mainly influenced by its intrinsic diffusivity across the SC, whereas variation in transport of CM could be attributed to both thermodynamic and kinetic differences among different anatomical skin sites.

In vitro/in vivo correlations between transdermal delivery of 5-aminolaevulinic acid and cutaneous protoporphyrin IX accumulation and effect of formulation

Summary Background Photodynamic therapy (PDT) using topical application of 5‐aminolaevulinic acid (ALA) has been widely reported for the treatment of a variety of neoplastic and non‐neoplastic cutaneous diseases. Although different formulations containing variable amounts of ALA have been applied in PDT, the dose–response relationships between transdermal ALA delivery and cutaneous protoporphyrin IX (PpIX) accumulation have not been studied.

Permeability barrier abnormality of hairless mouse epidermis after topical corticosteroid: characterization of stratum corneum lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography.

Topical corticosteroids (TCS) are among the most frequently used topical therapeutics. Recently, it has been shown that TCS not only has antiproliferative actions, but also inhibits the differentiation of the epidermis and finally perturbates stratum corneum (SC) barrier function. It is well established that epidermal barrier function resides within the intercellular lipids of the SC. However, to date, little is known about the effects of TCS on the structure and composition of SC lipids. We therefore used hairless mouse skin to study the sequential changes of the SC permeability barrier and their intercellular lipids by ruthenium tetroxide staining and high‐performance thin‐layer chromatography (HPTLC) during topical use of corticosteroids. The results demonstrated a progressive increase in transepidermal water loss accompanied by a diminution in the SC intercellular lipid lamellae, which showed a normal structure of individual lamella. Analysis of lipid composition by HPTLC after a 6‐week application of TCS also showed an obvious decrease in all the main components of SC lipids, which are known to constitute the permeability barrier of the skin. In light of these results, our work provides direct morphological evidence that TCS deteriorates the permeability barrier of epidermis when applied to normal skin.