Toxicology assessment of manganese oxide nanomaterials with enhanced electrochemical properties using human in vitro models representing different exposure routes.

In the present study, a comparative human toxicity assessment between newly developed Mn3O4 nanoparticles with enhanced electrochemical properties (GNA35) and their precursor material (Mn3O4) was performed, employing different in vitro cellular models representing main exposure routes (inhalation, intestinal and dermal contact), namely the human alveolar carcinoma epithelial cell line (A549), the human colorectal adenocarcinoma cell line (HT29), and the reconstructed 3D human epidermal model EpiDerm. The obtained results showed that Mn3O4 and GNA35 harbour similar morphological characteristics, whereas differences were observed in relation to their surface area and electrochemical properties. In regard to their toxicological properties, both nanomaterials induced ROS in the A549 and HT29 cell lines, while cell viability reduction was only observed in the A549 cells. Concerning their skin irritation potential, the studied nanomaterials did not cause a reduction of the skin tissue viability in the test conditions nor interleukin 1 alpha (IL- 1 α) release. Therefore, they can be considered as not irritant nanomaterials according to EU and Globally Harmonized System of Classification and Labelling Chemicals. Our findings provide new insights about the potential harmful effects of Mn3O4 nanomaterials with different properties, demonstrating that the hazard assessment using different human in vitro models is a critical aspect to increase the knowledge on their potential impact upon different exposure routes.

In Vitro and Clinical Evaluation of Cannabigerol (CBG) Produced via Yeast Biosynthesis: A Cannabinoid with a Broad Range of Anti-Inflammatory and Skin Health-Boosting Properties.

Cannabigerol (CBG) is a minor non-psychoactive cannabinoid present in Cannabis sativa L. (C. sativa) at low levels (<1% per dry weight) that serves as the direct precursor to both cannabidiol (CBD) and tetrahydrocannabinol (THC). Consequently, efforts to extract and purify CBG from C. sativa is both challenging and expensive. However, utilizing a novel yeast fermentation technology platform, minor cannabinoids such as CBG can be produced in a more sustainable, cost-effective, and timely process as compared to plant-based production. While CBD has been studied extensively, demonstrating several beneficial skin properties, there are a paucity of studies characterizing the activity of CBG in human skin. Therefore, our aim was to characterize and compare the in vitro activity profile of non-psychoactive CBG and CBD in skin and be the first group to test CBG clinically on human skin. Gene microarray analysis conducted using 3D human skin equivalents demonstrates that CBG regulates more genes than CBD, including several key skin targets. Human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEKs) were exposed in culture to pro-inflammatory inducers to trigger cytokine production and oxidative stress. Results demonstrate that CBG and CBD reduce reactive oxygen species levels in HDFs better than vitamin C. Moreover, CBG inhibits pro-inflammatory cytokine (Interleukin-1ß, -6, -8, tumor necrosis factor α) release from several inflammatory inducers, such as ultraviolet A (UVA), ultraviolet B (UVB), chemical, C. acnes, and in several instances does so more potently than CBD. A 20-subject vehicle-controlled clinical study was performed with 0.1% CBG serum and placebo applied topically for 2 weeks after sodium lauryl sulfate (SLS)-induced irritation. CBG serum showed statistically significant improvement above placebo for transepidermal water loss (TEWL) and reduction in the appearance of redness. Altogether, CBG's broad range of in vitro and clinical skin health-promoting activities demonstrates its strong potential as a safe, effective ingredient for topical use and suggests there are areas where it may be more effective than CBD.

Synthesis and biological evaluation of ruthenium polypyridine complexes with 18ß-glycyrrhetinic acid as antibacterial agents against Staphylococcus aureus.

Four new ruthenium(II) polypyridine complexes bearing 18ß-glycyrrhetinic acid derivatives, [Ru(bpy)2L](PF6)2 (Ru1), [Ru(dmb)2L](PF6)2 (Ru2), [Ru(dtb)2L](PF6)2 (Ru3) and [Ru(phen)2L](PF6)2 (Ru4) (bpy = 2,2-bipyridine, dmb = 4,4'-dimethyl-2,2'-bipyridine, dtb = 4,4'-di-tert-butyl-2,2'-bipyridine, phen = 1,10-phenanthroline and L is the GA modified new ligand) were designed and synthesized. Their antimicrobial activities against Staphylococcus aureus (S. aureus) were evaluated and all complexes showed an obvious inhibitory effect, especially, the minimum inhibitory concentration (MIC) value of Ru2 was 3.9 µg mL-1. Moreover, Ru2 was found to significantly inhibit the formation of biofilms. The membrane-compromising action mode was suggested to be their potential antibactericidal mechanism. In hemolysis experiments, Ru2 hardly showed cytotoxicity to mammalian erythrocytes. Furthermore, the synergism between Ru2 and common antibiotics, such as ampicillin, chloramphenicol, tetracyclines and ofloxacin, against S. aureus was also detected using the checkerboard method. Finally, a mouse skin infection model was established to evaluate the antibacterial activity of Ru2in vivo, and the results showed that Ru2 could effectively promote wound healing in mice infected with S. aureus. Moreover, the results of histopathological research were consistent with the results of the hemolysis test, indicating that the Ru2 complex was almost non-toxic. Thus, it was demonstrated that the polypyridine ruthenium complexes modified with glycyrrhetinic acid (GA) are a promising strategy for developing interesting antibacterial agents.

Topical Delivery of Geranium/Calendula Essential Oil-Entrapped Ethanolic Lipid Vesicular Cream to Combat Skin Aging.

The present study deals with the evaluation of the age-defying potential of topical cream formulations bearing Geranium essential oil/Calendula essential oil-entrapped ethanolic lipid vesicles (ELVs). Two types of cream formulations were prepared, viz., conventional and ELVs spiked o/w creams. Essential oil- (EO-) loaded ELVs were characterized by vesicle size, polydispersity index, encapsulation efficiency, and scanning electron microscopy. The cream formulations were evaluated for homogeneity, spreadability, viscosity, pH, in vitro antioxidant capacity, sun protection factor, and in vitro collagenase and elastase inhibition capacity. Confocal laser scanning microscopy (CLSM) was performed to ascertain skin permeation of conventional and vesicular cream. The results of in vitro antioxidant studies showed that GEO-/CEO-loaded vesicular creams have notable antioxidant capacity when compared to nonvesicular creams. GEO- or CEO-loaded vesicular creams exhibited the highest SPF value 10.26 and 18.54, respectively. Both the EO-based vesicular creams showed in vitro collagenase and elastase enzyme inhibition capacity. CLSM images clearly depicted that vesicular cream deep into the skin layers. From the research findings, the age-defying potential and photoprotective effects of GEO and CEO were confirmed. It can be concluded that ELVs are able to preserve the efficiency of EOs and have the potential to combat skin aging.

Ex Vivo Human Skin: An Alternative Test System for Skin Irritation and Corrosion Assays.

Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.

Nanoparticle impregnated self-supporting protein gel for enhanced reduction in oxidative stress: A molecular dynamics insight for lactoferrin-polyphenol interaction.

In the present work, lactoferrin (Lf) based nanoparticle incorporated self-supporting gel encapsulating a flavonoid, quercetin (Q), was developed. The complex formation between Lf and Q was assessed using molecular docking and dynamics simulation that lactoferrin and quercetin showed strong interaction and binding supporting hydrophobic interaction. The microscopic, spectroscopic, and x-ray techniques were used to characterize the gel extensively. In vitro drug release was studied to understand the release pattern of quercetin from the protein gel. The viscosity of the gel and its rheological characteristics were determined using a Brookfield viscometer. Ex vivo skin permeation studies using vertical diffusion cells were carried out to understand its skin permeation properties. The gel showed strong anti-oxidant activity using the DPPH scavenging assay. The enhanced effect of the Lf-Q complex on antioxidant enzyme activity (superoxide dismutase, catalase, and malondialdehyde), was supported by molecular dynamics, surface hydrophobicity, and in vitro studies. To investigate the effect of the gel on angiogenesis, the chorioallantoic membrane assay was performed and its compatibility with erythrocytes was also assessed. Suitability for topical administration was assessed using skin irritation studies performed on Sprague Dawley rats. The overall results suggest that the developed NiPG is suitable for cutaneous localization of quercetin with enhanced antioxidant activity.

Cause Clarification of Cysteine Oxidation by Active Species Generated during the Oxidation Process of Cinnamaldehyde and Impact on an In Chemico Alternative Method for Skin Sensitization Using a Nucleophilic Reagent Containing Cysteine.

Aldehydes comprise a major portion of skin sensitizers because they can react with both cysteine and lysine. Moreover, cinnamaldehyde (CA) is a typical moderate sensitizer and is often used in an alternative test method for skin sensitization. The amino acid derivative reactivity assay (ADRA) is an in chemico test method that evaluates the reactivity of cysteine derivatives (N-(2-(1-naphthyl)acetyl)-l-cysteine, NAC) and lysine derivatives with the test chemicals and uses CA as a proficiency substance. We found that NAC depletion for CA was only 10-20% when CA was used directly from the reagent bottle, although it increased to almost 100% when stored after being aliquoted from the reagent bottle. It was also found that this was due to the air oxidation of NAC itself rather than the reaction of NAC with CA, indicating that this result simply shows an increase in apparent reactivity. Aldehydes are known to produce active species, such as radicals, during air oxidation. Therefore, we investigated whether radicals were generated under storage conditions using the radical scavenger OH-TEMPO. LC/MS/MS analysis revealed that CA and OH-TEMPO complexes were produced during the air oxidation of CA. In the results of five aldehydes, similar to CA, active species were not generated as significantly as CA. Collectively, during the evaluation of the aldehydes, it can be seen that careful measures need to be taken to prevent the aldehydes from oxidizing during storage, indicating that assessment without preventing air oxidation carries an increased risk of overestimation compared with the intrinsic skin sensitization potency.

Skin Equivalent Models: Protocols for In Vitro Reconstruction for Dermal Toxicity Evaluation.

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.

Carbon dioxide inhibits UVB-induced inflammatory response by activating the proton-sensing receptor, GPR65, in human keratinocytes.

Carbon dioxide (CO2) is the predominant gas molecule emitted during aerobic respiration. Although CO2 can improve blood circulation in the skin via its vasodilatory effects, its effects on skin inflammation remain unclear. The present study aimed to examine the anti-inflammatory effects of CO2 in human keratinocytes and skin. Keratinocytes were cultured under 15% CO2, irradiated with ultraviolet B (UVB), and their inflammatory cytokine production was analyzed. Using multiphoton laser microscopy, the effect of CO2 on pH was observed by loading a three-dimensional (3D)-cultured epidermis with a high-CO2 concentration formulation. Finally, the effect of CO2 on UVB-induced erythema was confirmed. CO2 suppressed the UVB-induced production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) in keratinocytes and the 3D epidermis. Correcting medium acidification with NaOH inhibited the CO2-induced suppression of TNFα and IL-6 expression in keratinocytes. Moreover, the knockdown of H+-sensing G protein-coupled receptor 65 inhibited the CO2-induced suppression of inflammatory cytokine expression and NF-κB activation and reduced CO2-induced cyclic adenosine monophosphate production. Furthermore, the high-CO2 concentration formulation suppressed UVB-induced erythema in human skin. Hence, CO2 suppresses skin inflammation and can be employed as a potential therapeutic agent in restoring skin immune homeostasis.

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP-1, IL-8, IL-18 and IL-23 levels have a strong correlation with HS (P < .010-0.004; AUC = 0.790-0.883). Notably, combinations of two or three cytokines (TNF-a, MCP-1 and IL-23; AUC: 0.942, Nagelkerke R2 : 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.

Nanomaterial Lipid-Based Carrier for Non-Invasive Capsaicin Delivery; Manufacturing Scale-Up and Human Irritation Assessment.

Capsaicin is an active compound in chili peppers (Capsicum chinense) that has been approved for chronic pain treatment. The topical application of high-strength capsaicin has been proven to reduce pain; however, skin irritation is a major drawback. The aim of this study was to investigate an appropriate and scalable technique for preparing nanostructured lipid carriers (NLCs) containing 0.25% capsaicin from capsicum oleoresin (NLC_C) and to evaluate the irritation of human skin by chili-extract-loaded NLCs incorporated in a gel formulation (Gel NLC_C). High-shear homogenization with high intensity (10,000 rpm) was selected to create uniform nanoparticles with a size range from 106 to 156 nm. Both the NLC_C and Gel NLC_C formulations expressed greater physical and chemical stabilities than the free chili formulation. Release and porcine biopsy studies revealed the sustained drug release and significant permeation of the NLCs through the outer skin layer, distributing in the dermis better than the free compounds. Finally, the alleviation of irritation and the decrease in uncomfortable feelings following the application of the Gel NLC_C formulation were compared to the effects from a chili gel and a commercial product in thirty healthy volunteers. The chili-extract-loaded NLCs were shown to be applicable for the transdermal delivery of capsaicin whilst minimizing skin irritation, the major noncompliance cause of patients.

Anti-melanogenic effects of hot-water extracts from Torreya nucifera via MAPKs and cAMP signaling pathway on B16F10 cells.

Torreya nucifera is an evergreen tree in the family Taxaceae, the seeds, leaves, and stems of which have long been used as edible products and herbal medicines in Korea. Previous studies of biological activity have shown that T. nucifera has antioxidant and anti-inflammatory effects. However, the effect of T. nucifera leaves on melanogenesis are yet to be studied. In this investigation, we used B16F10 melanoma cells to test the efficacy of T. nucifera leaf hot water extract (TLWE). α-melanocyte stimulating hormone (α-MSH) stimulated B16F10 melanoma cells were treated with various concentrations of TLWE (50, 100, and 200 µg/mL). The results showed that TLWE reduced the melanin content and cellular tyrosinase activity in a concentration-dependent manner. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) signaling pathway. The compounds catechin and ρ-coumaric acid, which are known to have a whitening effect on skin, were detected by HPLC analysis. These results suggest that TLWE has an anti-melanogenic effect. In addition, the safety of TLWE was tested. The results of the skin irritation test showed that TLWE is harmless to the human skin, even at higher concentrations than those used in the experiment. Therefore, we suggest that the water extract of T. nucifera leaves has potential for use as a skin-whitening agent.

Evaluation of the sensitization potential of volatile and semi-volatile organic compounds using the direct peptide reactivity assay.

The purpose of this study was to evaluate the sensitization potential of 82 compounds classified as volatile and/or semi-volatile organic compounds using the direct peptide reactivity assay (DPRA), given that these chemical compounds have been detected frequently and at high concentrations in a national survey of Japanese indoor air pollution and other studies. The skin sensitization potential of 81 of these compounds was evaluable in our study; one compound co-eluted with cysteine peptide and was therefore not evaluable. Twenty-five of the evaluated compounds were classified as positive. Although all glycols and plasticizers detected frequently and at high concentrations in a national survey of Japanese indoor air pollution were negative, hexanal and nonanal, which are found in fragrances and building materials, tested positive. Monoethanolamine and 1,3-butanediol, which cause clinical contact dermatitis, and several compounds reported to have weak sensitization potential in animal studies, were classified as negative. Thus, it was considered that compounds with weak sensitization potential were evaluated as negative in the DPRA. Although the sensitization potential of the formaldehyde-releasing preservative bronopol has been attributed to the release of formaldehyde (a well-known contact allergen) by its degradation, its degradation products-bromonitromethane and 2-bromoethanol-were classified as positive, indicating that these degradation products also exhibit sensitization potential. The compounds that tested positive in this study should be comprehensively assessed through multiple toxicity and epidemiological studies.

Estudio del efecto citotóxico e irritativo de jabones para la limpieza cutánea / Study of the cytotoxic and irritant effects of skin cleansing soaps

Resumen Introducción: El jabón para el aseo cutáneo es de empleo común entre la población, sin embargo, es posible que cause daño a las células de la piel y modifique la barrera cutánea. Objetivo: Determinar el efecto citotóxico de los jabones en queratinocitos cultivados in vitro y correlacionarlo con la irritación clínica. Método: Se realizó una encuesta para conocer los jabones comerciales más utilizados y su cantidad; posteriormente, se evaluó su citotoxicidad en cultivos de queratinocitos humanos mediante el método de resazurina. Los jabones con mayor y menor citotoxicidad se aplicaron en piel de voluntarios sanos para evaluar su efecto en la barrera cutánea mediante ensayos de colorimetría y pérdida transepidérmica de agua. Resultados: De los jabones analizados, 37 % demostró ser tóxico para los queratinocitos in vitro. El jabón con mayor toxicidad indujo el mayor índice de eritema y pérdida transepidérmica de agua, en comparación con el jabón menos tóxico y el vehículo empleado como solución control. Conclusión: Los jabones comercializados para el aseo cutáneo pueden incluir ingredientes químicos que dañan los queratinocitos humanos y causan irritación subclínica de la barrera cutánea. Su utilización puede agravar dermatosis preexistentes, generar dermatitis xerósica o de contacto irritativa y causar atrofia y dermatoporosis.

Abstract Introduction: The use of soap for skin cleansing is common among the population. However, it is possible that it causes damage to skin cells and disrupts the skin barrier. Objective: To determine the cytotoxic effect of soaps on in vitro-cultured keratinocytes and to correlate it with clinical irritation. Method: A survey was conducted to find out the most widely used commercial soaps and their number. Subsequently, their cytotoxicity was evaluated in human keratinocyte cultures using the resazurin assay. The soaps with the highest and lowest cytotoxicity were applied to the skin of healthy volunteers to assess their effect on the skin barrier using colorimetry and transepidermal water loss (TEWL) assays. Results: Of the analyzed soaps, 37 % were shown to be toxic to keratinocytes in vitro. The soap with the highest toxicity induced the highest rate of erythema and TEWL, in comparison with the least toxic soap and the vehicle used as the control solution. Conclusion: Soaps marketed for skin cleansing can contain chemical ingredients that damage human keratinocytes and cause skin barrier subclinical irritation. Their use can worsen preexisting dermatoses, generate xerotic or irritant contact dermatitis, and cause atrophy and dermatoporosis.

Evaluation of skin irritation following weathered crude oil exposure in two mouse strains.

Petroleum crude oil spills are common and vary in size and scope. Spill response workers throughout the course of remediation are exposed to so-called weathered oil and are known to report diverse health effects, including contact dermatitis. A murine model of repeated exposure to weathered marine crude oil was employed utilizing two strains of mice, C57BL/6 and BALB/c, to investigate the pathology of this irritant and identify the principal hydrocarbon components deposited in skin. Histopathology demonstrated clear signs of irritation in oil-exposed skin from both mouse strains, characterized by prominent epidermal hyperplasia (acanthosis). BALB/c mice exposed to oil demonstrated more pronounced irritation compared with C57BL/6 mice, which was characterized by increased acanthosis as well as increased inflammatory cytokine/chemokine protein expression of IL-1ß, IL-6, CXCL10, CCL2, CCL3, CCL4, and CCL11. A gas chromatography/mass spectrometry method was developed for the identification and quantification of 42 aliphatic and EPA priority aromatic hydrocarbons from full thickness skin samples of C57BL/6 and BALB/c mice exposed to oil samples. Aromatic hydrocarbons were not detected in skin; however, aliphatic hydrocarbons in skin tended to accumulate with carbon numbers greater than C16. These preliminary data and observations suggest that weathered crude oil is a skin irritant and this may be related to specific hydrocarbon components, although immune phenotype appears to impact skin response as well.

Topical Application of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells in Combination with Sponge Spicules for Treatment of Photoaging.

PURPOSE: The topical application of exosomes secreted by mesenchymal stem cells (MSC-Exos) on the skin is a very new and interesting topic in the medical field. In this study, we aimed to investigate whether marine sponge Haliclona sp. spicules (SHSs) could effectively enhance the skin delivery of human umbilical cord-derived MSC-Exos (hucMSC-Exos), and further evaluate the topical application of hucMSC-Exos combined with SHSs in rejuvenating photoaged mouse skin. MATERIALS AND METHODS: SHSs were isolated from the explants of sponge Haliclona sp. with our proprietary method, and hucMSC-Exos were prepared from the conditioned medium of hucMSCs using ultracentrifugation. The effects of SHSs on the skin penetration of fluorescently labeled hucMSC-Exos were determined using confocal microscopy in vitro (porcine skin) and in vivo (mouse skin). The therapeutic effects of hucMSC-Exos coupled with SHSs against UV-induced photoaging in mice were assessed by using microwrinkles analysis, pathohistological examination and real-time RT-PCR. We also tested the skin irritation caused by the combination of hucMSC-Exos and SHSs in guinea pigs. RESULTS: In vitro results showed that hucMSC-Exos could not readily penetrate through porcine skin by themselves. However, SHSs increased the skin absorption of exosomes by a factor of 5.87 through creating microchannels. Similar penetration enhancement of hucMSC-Exos was observed after SHSs treatment in mice. The combined use of hucMSC-Exos and SHSs showed significant anti-photoaging effects in mice, including reducing microwrinkles, alleviating histopathological changes, and promoting the expression of extracellular matrix constituents, whereas hucMSC-Exos alone produced considerably weaker effects. Skin irritation test showed that the combination of hucMSC-Exos and SHSs caused slight irritation, and the skin recovered shortly. CONCLUSION: SHSs provide a safe and effective way to enhance the skin delivery of MSC-Exos. Moreover, the combination of MSC-Exos and SHSs may be of much use in the treatment of photoaging.

Novel Clove Essential Oil Nanoemulgel Tailored by Taguchi's Model and Scaffold-Based Nanofibers: Phytopharmaceuticals with Promising Potential as Cyclooxygenase-2 Inhibitors in External Inflammation.

PURPOSE: Clove essential oil is a phytochemical possessing a vast array of biological activities. Nevertheless, fabricating nano topical delivery systems targeted to augment the anti-inflammatory activity of the oil has not been investigated so far. Accordingly, in this study, controlled release nanoparticulate systems, namely nanoemulgel and nanofibers (NFs), of the oil were developed to achieve such goal. METHODS: The nanoemulsion was incorporated in the hydrogel matrix of mixed biopolymers - chitosan, guar gum and gum acacia - to formulate nanoemulsion-based nanoemulgel. Taguchi's model was adopted to evaluate the effect of independently controlled parameters, namely, the concentration of chitosan (X1), guar gum (X2), and gum acacia (X3) on different dependently measured parameters. Additionally, the nanoemulsion-based NFs were prepared by the electrospinning technique using polyvinyl alcohol (PVA) polymer. Extensive in vitro, ex vivo and in vivo evaluations of the aforementioned formulae were conducted. RESULTS: Both Fourier transform-infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) established the complete dispersion of the nanoemulsion in the polymeric matrices of the prepared nanoemulgel and NFs. The ex vivo skin permeation data of clove essential oil from the prepared formulations showed that NFs can sustain its penetration through the skin comparably with nanoemulgel. Topical treatment with NFs (once application) and nanoemulgel (twice application) evoked a marvelous in vivo anti-inflammatory activity against croton oil-induced mouse skin inflammation model when compared with pure clove essential oil along with relatively higher efficacy of medicated NFs than that of medicated nanoemulgel. Such prominent anti-inflammatory activity was affirmed by histopathological and immunohistochemical examinations. CONCLUSION: These results indicated that nanoemulsion-based nanoemulgel and nanoemulsion-based NFs could be introduced to the phytomedicine field as promising topical delivery systems for effective treatment of inflammatory diseases instead of nonsteroidal anti-inflammatory drugs that possess adverse effects.

Mitigation of Rheumatic Arthritis in a Rat Model via Transdermal Delivery of Dapoxetine HCl Amalgamated as a Nanoplatform: In vitro and in vivo Assessment.

PURPOSE: Dapoxetine HCl (DH), a selective serotonin reuptake inhibitor, may be useful for the treatment of rheumatic arthritis (RA). The purpose of this study was to investigate the therapeutic efficacy of transdermal delivery of DH in transethosome nanovesicles (TENVs). This novel delivery of DH may overcome the drawbacks associated with orally administered DH and improve patient compliance. METHODS: DH-TENV formulations were prepared using an injection- sonication method and optimized using a 33 Box-Behnken-design with Design Expert® software. The TENV formulations were assessed for entrapment efficiency (EE-%), vesicle size, zeta potential, in vitro DH release, and skin permeation. The tolerability of the optimized DH-TENV gel was investigated using a rat skin irritation test. A pharmacokinetic analysis of the optimized DH-TENV gel was also conducted in rats. Moreover, the anti-RA activity of the optimized DH-TENV gel was assessed based on the RA-specific marker anti-cyclic cirtullinated peptide antibody (anti-CCP), the cartilage destruction marker cartilage oligomeric matrix protein (COMP) and the inflammatory marker interleukin-6 (IL-6). Level of tissue receptor activator of nuclear factor kappa-Β ligand (RANKL) were also assessed. RESULTS: The optimized DH-TENV formulation involved spherical nanovesicles that had an appropriate EE- % and skin permeation characteristic. The DH-TENV gel was well tolerated by rats. The pharmacokinetics analysis showed that the optimized DH-TENV gel boosted the bioavailability of the DH by 2.42- and 4.16-fold compared to the oral DH solution and the control DH gel, respectively. Moreover, it significantly reduced the serum anti-CCP, COMP and IL-6 levels and decreased the RANKL levels. Furthermore, the DH-TENV gel attenuated histopathological changes by almost normalizing the articular surface and synovial fluid. CONCLUSION: The results indicate that DH-TENVs can improve transdermal delivery of DH and thereby alleviate RA.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

Oxidation of a cysteine-derived nucleophilic reagent by dimethyl sulfoxide in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 µm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.