Loss of elastic fibers causes skin wrinkles in sun-damaged human skin.

BACKGROUND: Although wrinkling is the most obvious sign of aged skin, the detailed pathomechanism of wrinkle development has not been elucidated. OBJECTIVES: In this study, we investigated the role of elastic fibers in the formation of skin wrinkles. METHODS: Loss of elastic fibers was measured quantitatively in the facial skins of subjects representing seven decades, and then compared with wrinkle severities. We also investigated whether topical retinoic acid treatment to photoaged human skin can restore destroyed elastic fiber, and the correlation between wrinkle improvement with increase in elastic fibers in RA-treated facial skin. RESULTS: We found a significant correlation between decreases in the length, width, number and total area of oxytalan fibers and wrinkle severity. Furthermore, we found that topical application of retinoic acid (0.025%) to chronically photodamaged skin regenerated and restored elastic fibers, and that there was a significant positive correlation between the amount of newly regenerated elastic fiber and the wrinkle improvement caused by retinoic acid. CONCLUSIONS: Our results provide an objective insight into the role of elastic fibers in skin wrinkle formation by providing a quantitative correlation between changes in oxytalan fibers and the severity of skin wrinkling.

Biflavonoids isolated from Selaginella tamariscina regulate the expression of matrix metalloproteinase in human skin fibroblasts.

The methanol extract from Selaginella tamariscina significantly inhibited UV irradiation induced activity of matrix metalloproteinase-1 (MMP-1) in primary fibroblasts from human skin. Using the technique of bioassay-directed chromatographic separation, five biflavonoids were isolated from the ethyl acetate soluble fraction of S. tamariscina. Here, we investigated the effect of these five biflavonoids on the regulation of MMP-1 and -2 in UV irradiated cultured dermal fibroblasts from human neonatal foreskins. Among these biflavonoids, sumaflavone and amentoflavone showed significant MMP-1 inhibitory activity in primary human dermal fibroblasts after UV irradiation. The IC(50) values of sumaflavone, amentoflavone and retinoic acid, which was used as a positive control, were 0.78, 1.8, and 10microM, respectively.

Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection.

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15-102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.

Suitability of macrophage inflammatory protein-1beta production by THP-1 cells in differentiating skin sensitizers from irritant chemicals.

BACKGROUND: Worldwide restrictions in animal use for research have driven efforts to develop alternative methods. OBJECTIVE: The study aimed to test the efficacy of the macrophage inflammatory protein-1beta (MIP-1beta) assay for testing chemicals' skin-sensitizing capacity. METHODS: The assay was performed using 9 chemicals judged to be sensitizing and 7 non-sensitizing by the standard in vivo assays. THP-1 cells were cultured in the presence or absence of 4 doses, 0.01x, 0.1x, 0.5x, or 1x IC(50) (50% inhibitory concentration for THP-1 cell proliferation) of these chemicals for 24 hr, and the MIP-1beta level in the supernatants was determined. Skin sensitization by the test chemicals was determined by MIP-1beta production rates. The MIP-1beta production rate was expressed as the relative increase in MIP-1beta production in response to chemical treatment compared with vehicle treatment. RESULTS AND CONCLUSION: When the threshold MIP-1beta production rate used was 100% or 105% of dimethyl sulfoxide, all the sensitizing chemicals tested (dinitrochlorobenzene, hexyl cinnamic aldehyde, eugenol, hydroquinone, dinitrofluorobenzene, benzocaine, nickel, chromium, and 5-chloro-2-methyl-4-isothiazolin-3-one) were positive, and all the non-sensitizing chemicals (methyl salicylate, benzalkonium chloride, lactic acid, isopropanol, and salicylic acid), with the exception of sodium lauryl sulfate, were negative for MIP-1beta production. These results indicate that MIP-1beta could be a biomarker for classification of chemicals as sensitizers or non-sensitizers.

Statistically designed enzymatic hydrolysis for optimized production of icariside II as a novel melanogenesis inhibitor.

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p<0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.

Determination and validation of six sunscreen agents in suncare products by UPLC and HPLC.

Methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine are sunscreen agents that have hydrophobic behaviors in common. They were not normally assayed with the following four sunscreen agents that have hydrophilic behaviors in a single chromatographic run: ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. For that reason, methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine require much time in order to assay products with those materials. A rapid, selective, and reproducible determination method needs to be developed for the simultaneous examination of methylene bis-benzotriazolyl tetramethyl butylphenol and bis-ethylhexyloxy phenol methoxyphenyl triazine with the sunscreen agents, ethylhexyl methoxycinnamate, isoamyl p-methoxycinnamate, ethylhexyl salicylate, and ethylhexyl triazone. This new technique could reduce time in examining the sunscreen agents and be effective for quality control of suncare products. In this paper, the HPLC and UPLC system is used for developing the determination of the sunscreen agents. Several evaluations of some mixtures of eluents and columns were obtained for the optimal condition of separation. In HPLC, the optimal peak resolution was obtained through ethanol-water gradient elution and a 75-mm C18 column with a 3.5-microm-sized particle on a flow rate of 1.0 ml/min. In UPLC, the most distinctive peak resolution was obtained through methanol-water gradient elution and a 50-mm C18 column with a 1.7-microm-sized particle on a flow rate 0.4 ml/min. Both of those chromatographic determination methods could be used in the examination of six types of sunscreen agents without any interference from other product excipients in the agents. The proposed determination methods were validated for specificity, linearity, repeatability, system stability, intermediate precision, and accuracy. Consequently, HPLC and UPLC determination methods could be rapid, selective, and proper applications for the assay of sunscreen agents in suncare products.

Simultaneous determination of heavy metals in cosmetic products.

An extremely small amount of several heavy metals have been detected in cosmetic products as impurities, which can cause skin allergies through percutaneous adsorption on the skin. We present here a fast, accurate, and highly sensitive method for simultaneous determination of Pb2+, Fe2+, Cu2+, Ni2+, Zn2+, Co2+, Cd2+ and Mn2+ in coloring agents and cosmetic products, to be evaluated by ion chromatography. All of these metals are well separated through a bifunctional ion-exchange column (IonPac CS5A) and detected by post-column reaction and spectrophotometric detection. The calibration graphs are linear (r2 > 0.999), in the range 0.1-1000 microg/ml. Detection limits for a 200-microl sample solution are at the mug/l level, which is sufficient for judging whether the product is safe or not. The relative standard deviations (RSDs) of the retention time and the peak area are less than 0.21% and 1.24%, respectively. The recovery rates are 97-104%. The result shows that the proposed determination method is more sensitive, more accurate, and faster than current methods such as HPLC, ICP-MS and Flame-AAS. The new method was applied to analyze the amount of heavy metals contained in 22 cosmetic products and 11 coloring agents.

Enhanced depigmenting effects of N-glycosylation inhibitors delivered by pH-sensitive liposomes into HM3KO melanoma cells.

Delivery activity of pH-sensitive 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):cholesteryl hemisuccinate (CHEMS) liposomes was assessed as an in vitro intracellular carrier system to increase the bioavailability of depigmentation actives. N-glycosylation inhibitors have a glycosylation-inhibiting effect, which is useful for the skin depigmentation that operates by interfering with the maturation of tyrosinase. However, an N-glycosylation inhibitor does not easily pass through skin or even cellular membranes due to its water-soluble property. Therefore, it should be transported to target cells by an efficient delivery carrier to reduce the glycosylated tyrosinase. Glycosylation-inhibiting and depigmentation effects of N-butyldeoxynojirimycine (NB-DNJ) and 1-deoxynojirimycine (DNJ)-loaded liposomes were evaluated using Western blotting and measurement of synthesized melanin. Interestingly, it was found that the pH-sensitive liposomes increased the glycosylation-inhibiting and thus, pigment-lightening effects of N-glycosylation inhibitors in vitro. In addition, cargo materials loaded in pH-sensitive liposomes were found to be much more efficiently delivered into the cytoplasm, as observed in fluorescent-activated cell sorting (FACS) and confocal laser-scanning microscopic (CLSM) analysis. These results indicate that pH-sensitive DOPE:CHEMS liposomes have a strong potential as a carrier system to promote delivery efficiency and to enhance the biological effects of water-soluble actives for applications in cosmetics, personal care products, and pharmaceutics.

Preparation of liposomes containing oleanolic acid via micelle-to-vesicle transition.

Micelle-to-vesicle transition method was used to make liposomes containing oleanolic acid. First, the solubilization of potassium salt of oleanolic acid at basic condition by micelle formation was confirmed. Using the soluble state of oleanolic acid at basic condition, liposomes containing oleanolic acid was prepared by adjusting pH. After making homogeneous aqueous mixture of potassium salt of oleanolic acid and lecithin in basic condition, the solution was neutralized to produce the lecithin-based liposomes that contain oleanolic acid inside the lipid bilayers. The optimal loading of oleanolic acid to lecithin (about 25 mole%) was found to exist to produce liposomal suspension of small size without homogenization step. Electron microscopy and dynamic light scattering studies showed that the narrowly distributed, reconstituted oleanolic acid-containing liposomes were prepared without severe mechanical treatment.

Effect of polymer characteristics on structure of polymer-liposome complexes.

In the current study, we examined the effect of polymer characteristics on the structure of complexes formed between poly(methacrylic acid-co-n-alkyl methacrylate) and with phosphatidylcholine/cholesterol liposomes. We varied the polymer concentration in the vesicles, the preparation concentration of lipid and polymer components during preparation, the molecular weight of the polymer chain, the molecular weight of the polymer's hydrophobic side groups and their mole fraction. The vesicle behavior indicated polymer-free bilayers and bilayers complexed with polymer coexisted at low polymer concentrations. As the polymer concentration exceeds a critical level, however, the system became homogeneous, indicating bilayer uniformity of the bilayer. As the polymer content was raised, the vesicle size and fluidity increased, and the transition temperature decreased. We found that the vesicle size mostly affects the membrane fluidity. We also found that the thermal properties (transition temperature and the magnitude of heat capacity of the peak, DeltaCp) are governed by the effects of the polymer on the structure of bilayer. The length of the alkyl chain of the polymer is shown to significantly affect the structure of polymer-liposome complexes, as did the chain molecular weight and mole concentration of hydrophobic group in the polymer.

The effects of Musk T on peroxisome proliferator-activated receptor [PPAR]-alpha activation, epidermal skin homeostasis and dermal hyaluronic acid synthesis.

Peroxisome proliferators activated receptors (PPARs) are a family of nuclear hormone receptors that heterodimer with the retinoid X receptor and function as transcriptional regulators of genes. Topically Applied PPAR-alpha agonists possess receptor mediated, pro-differentiating/anti-proliferative effects, lipid metabolism stimulation, and anti-inflammatory activity, which suggest that they could be beneficial for the treatment of a variety of cutaneous diseases. Hyaluronan (HA), a high-molecular-weight linear glycosaminoglycan consisting of alternating D: -glucuronic acid and N-acetyl-D: -glucosamine residues, is one of the major extracellular matrix components in skin. Among the family of HA synthase genes (HAS1, 2, 3) so far identified, one group has demonstrated that the expressions of HAS2 and HAS3 play crucial roles in the regulation of HA synthesis in human skin fibroblasts and keratinocytes, respectively, but the precise regulatory mechanisms are still unknown. We examine Musk T called Ethylene brassylate, Astratone or 1,4-Dioxacycloheptadecane-5,17-dione, which used as just a perfume ingredient, plays a role as PPAR-alpha ligand in vitro and stimulates skin barrier recovery, ceramide synthesis, beta-Glucocerebrosidase, involucrin expression in epidermis in vivo; and examine that Musk T stimulates HAS expression and HA synthesis in human skin fibroblast. Through these experiments, we conclude that Musk T is PPAR-alpha ligand, effects on keratinocyte differentiation, intercellular lipid synthesis in epidermis, HA synthesis stimulation in dermis.

Enzymatic preparation of kaempferol from green tea seed and its antioxidant activity.

Among the flavonols in green tea, kaempferol has many biological activities but kaempferol of plant origin is too expensive to be used in commercial products. Recently, we confirmed that green tea seed (GTS) contained a reasonable amount of kaempferol glycoside. After conducting structure analysis, two kaempferol glycosides were identified, kaempferol-3-O-[2-O-beta-D-galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-beta-D-xylopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside (compound 2), respectively. Also, a commercially useful method for kaempferol preparation was suggested by enzymatic hydrolysis using these two flavonoids. After several enzyme reactions were performed for the complete bioconversion of compounds 1 and 2 to kaempferol, we found that the optimum enzyme combination was reaction with beta-galactosidase and hesperidinase. Finally, we produced pure kaempferol with over 95% purity. We also compared the antioxidant effect of these two GTS flavonoids and its aglycone, kaempferol. Kaempferol is a more efficient scavenger of 1,1-diphenyl-2-picrylhydrazyl radicals and a better inhibitor of xanthine/xanthine oxidase than the two glycosides.

A novel volumetric method for quantitation of titanium dioxide in cosmetics.

Nowadays there are many sun-protection cosmetics incorporating organic or inorganic UV filters as active ingredients. Chemically stable inorganic sunscreen agents, usually metal oxides, are widely employed in high-SPF (sun protection factor) products. Titanium dioxide is one of the most frequently used inorganic UV filters. It has been used as a pigment for a long period of cosmetic history. With the development of micronization techniques, it has become possible to incorporate titanium dioxide in sunscreen formulations without the previous whitening effect, and hence its use in cosmetics has become an important research topic. However, there are very few works related to quantitation of titanium dioxide in sunscreen products. In this research, we analyzed the amounts of titanium dioxide in sunscreen cosmetics by adapting redox titration, reduction of Ti(IV) to Ti(III), and reoxidation to Ti(IV). After calcification of other organic ingredients of cosmetics, titanium dioxide is dissolved by hot sulfuric acid. The dissolved Ti(IV) is reduced to Ti(III) by adding metallic aluminum. The reduced Ti(III) is titrated against a standard oxidizing agent, Fe(III) (ammonium iron(III) sulfate), with potassium thiocyanate as an indicator. In order to test the accuracy and applicability of the proposed method, we analyzed the amounts of titanium dioxide in four types of sunscreen cosmetics, namely cream, make-up base, foundation, and powder, after adding known amounts of titanium dioxide (1 approximately 25 w/w%). The percentages of titanium dioxide recovered in the four types of formulations were in the range between 96% and 105%. We also analyzed seven commercial cosmetic products labeled with titanium dioxide as an ingredient and compared the results with those obtained from ICP-AES (inductively coupled plasma-atomic emission spectrometry), one of the most powerful atomic analysis techniques. The results showed that the titrated amounts were well in accord with the analyzed amounts of titanium dioxide by ICP-AES. Although instrument-based analytical methods, namely ICP-MS (inductively coupled plasma-mass spectrometry) and ICP-AES, are best for the analysis of titanium, it is difficult for small cosmetic companies to install such instruments because of their high cost. It was found that the volumetric method presented here gives quantitatively accurate and reliable results with routine lab-ware and chemicals.

The effects of a novel synthetic retinoid, seletinoid G, on the expression of extracellular matrix proteins in aged human skin in vivo.

BACKGROUND: Although retinoids have potential efficacy in aged skin, their side effect (skin irritation) remains a clinical problem. We designed a novel synthetic retinoid, seletinoid G, by using computer-aided molecular modeling, and investigated its effects on the expression of extracellular matrix proteins in human skin in vivo. METHODS: Twenty-three subjects were tested on the buttocks using 4-day occlusive application of seletinoid G and all-trans retinoic acid (tRA). Skin irritation after topical application was quantified by the degree of erythema and cutaneous blood flow. The expression of extracellular matrix proteins and interstitial collagenase (MMP-1) in skin biopsies was investigated by immunohistochemical staining and Western blotting. RESULTS: The topical application of seletinoid G under occlusion induced no skin irritation in contrast to tRA, which caused severe erythema. The topical treatment with seletinoid G increased the expressions of type I procollagen, tropoelastin, and fibrillin-1, and reduced MMP-1 in old skin in vivo. Seletinoid G was found to inhibit not only the UV-induced decrease of type I procollagen but the UV-induced increase of MMP-1 and c-Jun protein in young skin in vivo. CONCLUSIONS: Seletinoid G is a novel synthetic retinoid, which has little the side effect of skin irritation after topical application. Seletinoid G can repair altered connective tissue in old skin and inhibit UV-induced collagen deficiency in young skin.

New micelle-like polymer aggregates made from PEI-PLGA diblock copolymers: micellar characteristics and cellular uptake.

New amphiphilic block copolymers based on oligomeric polyethylenimine and poly(D,L-lactide-co-glycolide) (PEI-PLGA) were synthesized by directly coupling PLGA with a carboxyl terminal group to PEI. The block copolymers were prepared by varying the length of the hydrophobic PLGA block (M(n)=6, 10, and 21K), while that of the hydrophilic PEI block (M(n)=423) was fixed. PEI-PLGA block copolymers were found to be self-assembled in water by using a PLGA segment as a hydrophobic aggregate block and a PEI segment as a hydrophilic corona-forming block. The block copolymers formed micelle-like aggregates with critical association concentration (cac) in the range of 1.54-2.57x10(-3)g/l in water. It was found that the size and cac of the aggregates depended on the hydrophobic block length and the ionic state of the PEI block. The aggregate size decreased and the cac increased, when the PLGA block length decreased and the PEI block was protonated. As a general program aimed at the development of a new nanoscopic drug carrier, the cellular uptake behavior of PEI-PLGA aggregates was compared with that of plain PLGA nanoparticles by using confocal microscopy. The results showed that PEI-PLGA aggregates was readily adsorbed onto the cell surfaces and translocated into the cytoplasm, implying their versatile applicability as a drug carrier.

The extract of Thujae occidentalis semen inhibited 5alpha-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse.

BACKGROUND: The conversion of testosterone to dihydrotestosterone; 5alpha-androstan-17beta-ol-3-one by 5alpha-reductase plays a crucial role in hair baldness and prostatomegaly. Recent approach showed specific inhibitors for 5alpha-reductase type 2 such as finasteride promoted hair growth in male pattern alopecia. OBJECTIVE: In order to search for effective medicinal plant extracts applied topically for androgenetic alopecia, we screened natural plant extracts having inhibitory activities of 5alpha-reductase type 2 and demonstrated its biological function in androgen-related animal models. METHODS: We evaluated the inhibition activities of numerous plant extracts by contact cell based metabolic method using a stable HEK 293 cell line expressing human 5alpha-reductase (type 2). To elucidate the biological activity in vivo, the Thujae occidentalis semen (TOS) extract was topically applied to fuzzy rat and androchronogenetic alopecia (AGA) mouse, respectively. The secreted sebum and the size of sebaceous glands of fuzzy rat were measured after 6 weeks. Also, after the topical treatment with TOS extract and androgen receptor antagonist (cyproterone acetate) simultaneously with subcutaneous injection of testosterone (1 mg/mice/day), hair loss patterns of female B6CBAF1/j hybrid mouse were observed. RESULTS: TOS extract showed higher inhibition activity of 5alpha-reductase type 2(IC(50) value=2.6 microg/ml) than that of gamma-linolenic acid, but lower than that of finasteride. When applied to fuzzy rat, the amount of sebum and sebaceous gland size decreased remarkably. In AGA model, alopecia degrees of two groups, treated with TOS extract (P<0.015) or cyproterone acetate (P<0.01), were lower than that of vehicle (propylene glycol:ethanol=7:3) and there was no difference between above two groups. CONCLUSION: We have demonstrated the inhibitory activity of TOS extract for 5alpha-reductase type 2 and its biological action in two animal models, suggesting that TOS extract would be used as an effective agent for male pattern baldness by modifying androgen conversion.

Self-aggregates of poly(2-hydroxyethyl aspartamide) copolymers loaded with methotrexate by physical and chemical entrapments.

Amphiphilic copolymers based on poly(2-hydroxyethyl aspartamide) (PHEA) formed self-aggregates for the entrapment and release of methotrexate (MTX) by physical entrapment and chemical conjugation. In physical entrapment, MTX was partitioned into hydrophobic domains in self-aggregates of PHEA grafted with octadecyl chains (PHEA-C18) and the amount of the entrapped drug increased linearly by 3.39 mg per the degree of substitution of grafted octadecyl groups. The amphiphilic nature of the drug induced a large initial release in the buffer medium, irrespective of the amount of octadecyl chains. However, PEG-grafted PHEA-C18 copolymers conjugated with MTX, ConG, formed a micelle-like structure by self-association of the conjugates and suppressed the initial large release. The alkyl grafting lowered the CAC, meaning enhancement of aqueous stability. The release was accelerated in pH 10.0 by rapid hydrolysis of ester linkage by base-catalyzed cleavage, while it was significantly reduced at pH 5.0.

Transdermal delivery of mixnoxidil with block copolymer nanoparticles.

We evaluated the effect of hydrodynamic size of self-assembled nanoparticles on skin penetration of minoxidil in vitro and in vivo. Self-assembled 40- and 130-nm nanoparticles, both containing minoxidil, were prepared by solvent evaporation of poly(-caprolactone)-block-poly(ethyleneglycol) and were applied onto the skin of both hairy and hairless guinea pigs in the Franz diffusion cell. In hairy guinea pig skin, the permeation of the minoxidil that incorporated in 40-nm nanoparticles was 1.5-fold higher in the epidermal layer and 1.7-fold higher in the receptor solution than that of 130-nm nanoparticles. Nanoparticle size dependence on the permeation behavior of minoxidil was not observed for hairless guinea pig skin in either the epidermal layer or the receptor solution. Phospholipid liposomes and ethanol-water admixture, on the other hand, containing the same amount of minoxidil did not show differences in the amount of permeation irrespective of the existence of hair follicles. Confocal microscopy coupled with in vivo and in vitro skin permeation results demonstrated that nanoparticles containing solutes penetrated mainly via shunt routes like hair follicles, resulting in skin absorption of solutes.

Determination of zeta potentials of polymeric nanoparticles by the conductivity variation method.

An easy method of measurement of the zeta potentials of sub-50-nm polymeric nanoparticles is suggested. Although zeta potential measurements of nanoparticles or emulsions above 50 nm have been successfully carried out, zeta potentials of emulsions or nanoparticles below 50 nm could not be precisely measured in the region of extremely low conductivity by conventional electrophoresis with a He-Ne laser. However, zeta potentials of sub-50-nm nanoparticles were measured in the region of thin electrical double layers by addition of sodium chloride and zeta potentials in the condition without sodium chloride could be predicted by extrapolation of the measured potentials. The electrophoretic mobility of 150-nm nanoparticles stabilized with sodium dodecylsulfate was the same as that calculated from extrapolation of the measured ones. The zeta potentials of sub-50-nm nanoparticles stabilized with sodium dodecylsulfate could be calculated by the same procedure.

The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-water double emulsion by controlling pH and electrolyte concentration.

This study presents a new approach that can stabilize effectively L-ascorbic acid in water-in-oil-in-water (w/o/w) double emulsions. Basically, the behavior of L-ascorbic acid in the aqueous phase was observed, considering its molecular deformation. Then, it was found that the stability determined in the aqueous phase by high-performance liquid chromatography (HPLC) showed that the collapse of ionization of L-ascorbic acid played a crucial role in protecting the molecular deformation. Then, the stable aqueous system was incorporated into the internal aqueous phase of the double emulsions. From the HPLC analysis, it was observed that the L-ascorbic acid in an appropriate system showed high molecular stability for a long time. Moreover, in the measurement of in vitro skin permeation, the L-ascorbic acid stabilized in this study showed considerable skin permeation ability, indicating its potential applicability in pharmaceutics and cosmetics.