Primary Epidermal Keratinocytes Research Articles

A survey of multiple candidate probiotic bacteria reveals specificity in the ability to modify the effects of key wound pathogens.

We have evaluated the inhibitory effects of supernatants and lysates derived from several candidate probiotics, on the growth and biofilm formation of wound pathogens, and their ability to protect human primary epidermal keratinocytes from the toxic effects of pathogens. Supernatants (neutralized and non-neutralized) and lysates (via sonication) from Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Bifidobacterium longum, Lacticaseibacillus rhamnosus GG, and Escherichia coli Nissle 1917 were tested for their inhibitory effects against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumanni. The supernatants of L. plantarum, L. rhamnosus, B. longum, and L. rhamnosus GG reduced the growth of S. aureus, E. coli, and A. baumanni. B. longum additionally inhibited P. aeruginosa growth. However, neutralized Lactobacillus supernatants did not inhibit growth and in some cases were stimulatory. Lysates of L. plantarum and L. reuteri inhibited S. pyogenes while B. longum lysates inhibited E. coli and S. aureus growth. E. coli Nissle 1917 lysates enhanced the growth of S. pyogenes and P. aeruginosa. Biofilm formation by E. coli was reduced by lysates of L. reuteri and neutralized supernatants of all candidate probiotics. P. aeruginosa biofilm formation was reduced by E. coli Nissle supernatant but increased by L. plantarum, L. reuteri, and Bifidobacterium longum lysates. L. reuteri decreased the toxic effects of S. aureus on keratinocytes while E. coli Nissle 1917 lysates protected keratinocytes from S. pyogenes toxicity. In conclusion, lactobacilli and E. coli Nissle lysates confer inhibitory effects on pathogenic growth independently of acidification and may beneficially alter the outcome of interactions between host cell-pathogen in a species-specific manner.IMPORTANCEOne of the attributes of probiotics is their ability to inhibit pathogens. For this reason, many lactobacilli have been investigated for their effects as potential topical therapeutics against skin pathogens. However, this field is in its infancy. Even though probiotics are known to be safe when taken orally, the potential safety concerns when applied to potentially compromised skin are unknown. For this reason, we believe that extracts of probiotics will offer advantages over the use of live bacteria. In this study, we have surveyed five candidate probiotics, when used as extracts, in terms of their effects against common wound pathogens. Our data demonstrate that some probiotic extracts promote the growth of pathogens and highlight the need for careful selection of species and strains when probiotics are to be used topically.

The cytotoxic and immunomodulatory mechanisms of photodynamic therapy evaluated in in vitro studies on cultures of selected skin cell lines

SignificanceThe aim of the study is to evaluate the cytotoxic and immunomodulatory effects of HYP-PDT in vitro studies on cultures of selected skin cell lines, as a possibility of using PDT for patients with atopic dermatitis and psoriasis. ApproachPrimary Dermal Fibroblast and Primary Epidermal Keratinocytes was used in the study. After incubation the cells were treated with hypericin, exposed to VIS visible light. Then cell viability was determined by the MTT assay and cytokines markings were done with Bio-Plex Pro™ Assay kit and with Bio-Plex Suspension Array System. ResultsThe experiment showed a significant cytotoxic effect of HYP-PDT and in sublethal doses, an immunomodulatory effect manifested in changes in the concentrations of selected cytokines ConclusionsThe obtaining results confirming the destructive effect of HYP- PDT on the selected, skin cells lines show a possibility of extending the indication for HYP-PDT, for patients with inflammatory skin diseases.

Abstract B035: Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes

Abstract DNA damage associated with ultraviolet (UV) radiation exposure causes accelerated skin aging and cancer-initiating mutations. Over-the-counter UVA and UVB filters are required in all sunscreens, but transdermal absorption of these compounds have been reported to impair development and reproduction and metabolize into carcinogenic/mutagenic chemical intermediates. To assess the effects of sunscreen active ingredients on UVR-induced DNA damage and repair, we developed a novel high throughput (HTP) screening platform, UValidate, employing mixed populations of isogenic skin cells, exposed to single or combinations of UV filters in the presence of UVB and UVA. UValidate contains a patented LED UVR DNA damage induction system (LUDIS:V3), facilitating programmable precise induction of DNA damage and repair at multiple time points and replicates, using a 96-well configuration capable of simultaneous delivering UVA and UVB. LUDIS:V3 optimization experiments surprisingly revealed that, unlike keratinocytes, skin fibroblasts only repaired UVA DNA damage in conditioned media. We then tested sunscreen active ingredient combinations for their capacity to alter DNA repair and found that: 1) Some sunscreen ingredients, including dioxybenzone, induce a significant increase in DNA damage above endogenous levels. 2) Fibroblasts and keratinocytes respond differently to the treatment protocols. 3) Some sunscreen active compounds contribute to reduction of UVA-induced DNA damage. 4) No sunscreen completely inhibits all UVA DNA damage. We are currently employing the LUDIS:V3 system with HTP comet assays to compare DNA damage and DNA repair capacities, +/- sunscreen active ingredients, of six isogenic sets of primary epidermal keratinocytes (NHEK), melanocytes (NHEM), and fibroblasts (NHDF)` with different Fitzpatrick phototypes I-VI, representative of the diverse American population. These cells were isolated from donor skin samples, and further purified to establish banks of isogenic NHEK, NHEM, and NHDF cell lines. UVA exposure with the LUDIS:V3 and comet assays revealed higher levels of DNA damage in NHEK and NHDF, compared to NHEM, likely due to the UVR-protective effects of melanin in NHEM. Consistently, NHEK with phototype V (from darkly pigmented skin with higher melanin levels) exhibit significantly less UVA-induced DNA damage compared with phototype II NHEK. To create cellular models of dermal disease for DNA repair assays, we have successfully knocked out XPA in multiple NHDF cell lines with different phototypes using a CRISPR-Cas9 KO strategy, as confirmed by immunoblot and NGS sequence analyses. These and other engineered DNA repair-deficient skin cells will be assayed as single or mixed cell-type 2D cultures, with fluorescent labeling allowing for identification of subpopulations in comet and alkaline diffusion assays. This technology addresses the need for better platforms for rapid genotoxicity testing of safer and more effective chemical UV filters to prevent cutaneous carcinogenesis in diverse populations. Citation Format: Dean S. Rosenthal, Elijah Finn, Devin Teehan, Nusrat Islam, Veerupaxagouda Patil, Bonnie Carney, Scott S. Rosenthal, Lucia Dussan, Cynthia M. Simbulan-Rosenthal, Peter Sykora. Developing the UValidate platform to measure DNA damage and repair capacity in isogenic donor-derived skin keratinocytes, fibroblasts and melanocyte cell-lines with different Fitzpatrick phototypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B035.

EVALUATION OF THE CYTOTOXICITY AND GENOTOXICOLOGICAL SAFETY PROFILE OF BIOACTIVE SILVER(I) COMPLEXES WITH AMINOADAMANTANE LIGANDS

Silver(I) complexes of amantadine (atdH), memantine (mtnH) and rimantadine (rtdH), named Ag-atd, Ag-mtn and Ag-rtd, respectively, were recently synthesized and described in the literature as promising antibacterial agents. In the present study, the cytotoxicity of such complexes was evaluated against cultures of primary epidermal keratinocytes (HaCaT) and murine melanoma tumor cells (B16-F10), and mutagenicity was studied by the Ames test to investigate their abilities to induce gene mutations. The Ames test was performed using Salmonella Typhimurium strains (TA98, TA100, TA102 and TA97a) capable of detecting frameshift and base pair substitution gene mutations, in experiments with and without metabolic activation (microsomal fraction S9). This study revealed significant cytotoxic activity against tumor cells and selectivity of Ag-atd and Ag-rtd complexes when compared to non-tumor human keratinocyte cells. Moreover, the Ag(I) complexes did not induce a significant growth in the number of revertant colonies when comparing with the negative control, both in the experiments without and with metabolic activation, indicating absence of mutagenic activity. The results are encouraging and collaborate in the genotoxicological investigations necessary for understanding the interaction and ability of the silver complexes to induce mutations and contribute to ensure their uses as future antibacterial or antitumor agents.

Effective Healing of Staphylococcus aureus-Infected Wounds in Pig Cathelicidin Protegrin-1-Overexpressing Transgenic Mice.

Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing.

IL-17A-targeting fenofibrate attenuates inflammation in psoriasis by inducing autophagy

IL-17A is a critical pro-inflammatory cytokine in autoimmune diseases such as psoriasis. Targeting of IL-17A is an effective strategy to treat patients with autoimmune diseases; however, relevant small molecule therapeutics have not yet been developed. Here, the small molecule drug fenofibrate was validated as an inhibitor of IL-17A through ELISA and surface plasmon resonance (SPR) assays. We further confirmed that fenofibrate blocked IL-17A signalings including the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, in IL-17A-treated HaCaT cells, HEKa (human primary epidermal keratinocytes) and imiquimod (IMQ)-induced psoriasis mouse model. Fenofibrate attenuated systemic inflammation by suppressing Th17 populations and inflammatory cytokines, such as IL-1β, IL-6, IL-17A, and tumor necrosis factor (TNF). Surprisingly, fenofibrate upregulated LC3 and p62 in the psoriatic mouse group. The autophagy changes were caused by ULK1 pathway in hIL-17A-treated HaCaT and HEKa. In addition, the enhancement of autophagy by fenofibrate exerted anti-inflammatory effects, as demonstrated by the suppression of IL-6 and IL-8 in the IL-17A-treated keratinocytes. Thus, IL-17A-targeting fenofibrate can be a potential therapeutic for psoriasis and other autoimmune diseases via regulating autophagy.

Bioactivity and molecular docking of lactones isolated from Centaurea pseudosinaica Czerep

Two cytotoxic sesquiterpene lactones, 17-epichlorohyssopifolin A (1) and chlorjanerin (2), and a monoterpene lactone, loliolide (3) were isolated from Centaurea pseudosinaica. The cytotoxicity of the total extract and terpenoids 1–3 were evaluated against three human cancer cells (HepG2, PC-3, and HT-29), along with the human normal primary epidermal keratinocytes (HEKa) cells. With IC50 values ranging between 0.6 ± 0.04 and 5.0 ± 0.61 μg/mL against HepG2; 0.2 ± 0.01 and 11.9 ± 1.31 μg/mL against PC-3, and 0.04 ± 0.013 and 8.9 ± 0.97 μg/mL against HT-29, the total extract, and lactones 1–3 demonstrated cytotoxic effects. Compound 1 displayed the strongest impact on all cancer cells and a slightly safe effect on the normal cells HEKa. Compound 1 caused accumulation of HepG2 and HT-29 cells in G1 phase as displayed cell cycle analysis. On the other hand, the cell distributions were increased in the S phase in PC-3 cells. Furthermore, 1 caused apoptosis in PC-3 and HePG2 cells with 91.50%, and 79.72 %, respectively. A higher fraction of necrotic cells was observed in HT-29 cells amounting to 23.60%. These results suggested that the promising cytotoxicity exhibited by 1 is brought by the apoptosis induction in the cancer cells, which were evaluated. As the compounds showed antiproliferative effect against the HT-29 cells, the docking simulation was performed aiming at determining how they would interact with the EGFR enzyme, whose PDB: 4I23 is considered one of the two distinct wild types of EGFR enzymes. The antibacterial activity results revealed that 3 showed the most remarkable antibacterial effects, especially against the examined Gram-positive bacteria. The total extract exhibited potent activity against all examined bacteria. The total extract showed a potent antifungal effect against two Candida and two Aspergillus pathogens. The antioxidant activity revealed the potency of the total extract and 3 as antioxidant candidates. The obtained results refer to the importance of Centaurea pseudosinaica as a source of potent antiproliferative agents and the whole plant as an antipathogenic and antioxidant agent.

SG-APSIC1094: Impact of environmental pollution on skin antimicrobial peptide genes expression revealed by transcriptome profiling

Objectives: Pollution exposure is associated with several dermatological conditions including acne, atopic dermatitis, and psoriasis. Antimicrobial peptides (AMPs) are key effectors of innate defense, and some AMPs are involved in inflammatory skin conditions. In this study, we aimed to characterize expression changes of human AMPs under different in-vitro pollution exposures. Methods: RNA-seq profiling was conducted on normal human primary epidermal keratinocytes (NHEK) treated with either a vehicle control, or benzo[a]pyrene (BaP) and on pigmented living skin equivalent models (pLSE) treated with either a vehicle control, ozone, or vehicle exhaust. Differential expressed genes (DEGs) were identified with R scripts. DEGs of PM2.5 were obtained from the literature and the GEO database. Also, 180 human AMP genes were obtained from a UDAMP database. UpSetPlot was used to plot DEGs overlaps. MetaVolcano was used to identify frequently changed AMPs. Results: We used in-house and published transcriptome profiles to identify AMP genes that displayed altered expression under in-vitro pollution exposure. Of the 180 AMP genes under investigation, 37 showed significant changes in expression in at least 1 of the 5 experiments. Using MetaVolcano, 13 AMP genes were identified to be frequently and consistently changed. Several AMPs associated with inflammation and skin diseases were frequently upregulated, including S100A8, S100A9, LCN2, HBD3, RNASE7, and CXCL1. Only 3 frequently downregulated AMP genes were identified, including CXCL14, which is reported to be a noninflammatory AMP that is highly expressed in healthy skin and is downregulated in skin diseases. Conclusions: The data sets suggest that expression of both proinflammatory and homeostatic AMPs can be perturbed by pollution. These findings provide new clues to explain how pollution affects skin innate defense, host–microbe interactions and contributes to abnormal skin conditions. Normalizing aberrant AMP expression may be a potential approach to treat pollution associated skin disorders in the future.

Fibroblast-Derived Extracellular Vesicle-Packaged Long Noncoding RNA Upregulated in Diabetic Skin Enhances Keratinocyte MMP-9 Expression and Delays Diabetic Wound Healing

Accurate communication between fibroblasts and keratinocytes is crucial for diabetic wound healing. Extracellular vesicles are being explored as essential mediators of intercellular communication in the skin. However, the mechanisms underlying wound healing mediated by fibroblast-derived extracellular vesicles (Fib-EVs) remain unclear. The present study evaluated the role of long noncoding RNA upregulated in diabetic skin (lnc-URIDS) packed in Fib-EVs in the wound healing of streptozotocin-induced diabetes and the potential mechanisms of the effects. We demonstrated that high glucose induced the enrichment of lnc-URIDS in Fib-EVs, facilitated the transfer of lnc-URIDS to primary rat epidermal keratinocytes, and increased the expression of matrix metalloproteinase-9. Mechanistically, the binding of lnc-URIDS to YTH domain family protein-2 enhanced the degradation of YTH domain family protein-2 in the lysosomes, which increased the translational activity of the messenger RNA of matrix metalloproteinase-9 and ultimately induced the degradation of collagen for wound healing. The results provided an insight into the crosstalk and cooperation between fibroblasts and keratinocytes in collagen homeostasis in diabetic wounds and clarified the mechanism by which lnc-URIDS degrades collagen for diabetic wound healing.

Scutellaria baicalensis Georgi regulates REV-ERBα/BMAL1 to protect against skin aging in mice.

Scutellaria baicalensis Georgi (SBG) is a traditional Chinese medicine widely used to treat disorders such as hypertension, dysentery and hemorrhaging. Here, we aimed to assess the pharmacological effects of SBG on skin aging and to investigate the underlying mechanisms. Mice with skin aging were established by treatment with D-galactose and ultraviolet-B. SBG (topical application) showed a protective effect on skin aging in mice, as evidenced by less formation of skin wrinkles, higher levels of SOD (superoxide dismutase) and HYP (hydroxyproline) as well as a lower level of MDA (malondialdehyde). In the meantime, skin MMP-1 and p53 expression were lower, epidermis was thinner and collagen amount was higher in SBG-treated mice. Anti-skin aging effects of SBG were also confirmed in NIH3T3 and HaCaT cells, as well as in mouse primary dermal fibroblasts and human primary epidermal keratinocytes. Furthermore, we found that loss of Rev-erbα (a known repressor of Bmal1) up-regulated skin BMAL1 (a clock component and a known anti-aging factor) and ameliorated skin aging in mice. Moreover, SBG dose-dependently increased the expression of BMAL1 in the skin of aged mice and in senescent NIT3H3 cells. In addition, based on a combination of Gal4 chimeric, luciferase reporter and expression assays, SBG was identified as an antagonist of REV-ERBα and thus an inducer of BMAL1 expression. In conclusion, SBG antagonizes REV-ERBα to up-regulate BMAL1 and to protect against skin aging in mice.

The cannabinoid receptors system in horses: Tissue distribution and cellular identification in skin.

BackgroundThe endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid‐based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become disordered in pathological conditions. Cannabinoid receptors may be a possible therapeutic target in skin diseases.ObjectivesTo characterize the distribution and cellular expression of CBRs in the skin of horses under normal conditions.AnimalsFifteen healthy horses.MethodsUsing full‐thickness skin punch biopsy samples, skin‐derived primary epidermal keratinocytes and dermal‐derived cells, we performed analysis of Cnr1 and Cnr2 genes using real‐time PCR and CBR1 and CBR2 protein expression by confocal microscopy and Western blotting.ResultsNormal equine skin, including equine epidermal keratinocytes and dermal fibroblast‐like cells, all exhibited constant gene and protein expression of CBRs.Conclusions and Clinical ImportanceOur results represent a starting point for developing and translating new veterinary medicine‐based pharmacotherapies using ECS as a possible target.

IL-33 Enhances ACE2 Expression on Epidermal Keratinocytes in Atopic Dermatitis: A Plausible Issue for SARS-CoV-2 Transmission in Inflamed Atopic Skin.

Background: Interleukin-33 (IL-33) is an important cytokine in the pathophysiology of atopic dermatitis (AD) and in the progression of COVID-19. Angiotensin converting enzyme 2 (ACE2), the entry receptor for SARS-CoV-2, is expressed in epidermal keratinocytes. Whether IL-33 could regulate the expression of ACE2 mechanistically in keratinocytes warrants investigation. Objective: We questioned whether the ACE2 expression is increased in AD skin. We also questioned whether ACE2 is expressed in keratinocytes; if so, would its expression be enhanced mechanistically by IL-33. Methods: We measured and compared the expression of ACE2 in skin from patients with AD, patients with psoriasis, and healthy controls using immunohistochemistry. Flow cytometry, immunofluorescent exam, and quantitative RT-PCR were used for measuring the ACE2 expression in cultured keratinocytes treated with IL-33 and IL-17. Blocking antibodies were utilized to study the intracellular signaling pathways governing the ACE2 expression using cytokines. Results: The results showed that the ACE2 expression is increased in AD compared with that in healthy skin and psoriasis. In primary epidermal keratinocytes, ACE2 is constitutively expressed. IL-33 induces a time-dependent increase in ACE2 expression in cultured keratinocytes through quantitative PCR, flow cytometry, and immunofluorescent examinations. Furthermore, pretreatment of an ERK inhibitor, but not a STAT3 inhibitor, eliminated the increases in ACE2 by IL-33 in keratinocytes, indicating that IL-33 enhances ACE2 expression through ERK on epidermal keratinocytes. Conclusion: This is the first study to reveal that IL-33 enhances ACE2 expression on keratinocytes via ERK. Although further mechanistic studies are required, the increased ACE2 expression in IL-33 might have a biological implication on the transmission of SARS-CoV-2 in patients with AD.

Mechanical Stretch Triggers Epithelial-Mesenchymal Transition in Keratinocytes Through Piezo1 Channel

The epithelial-mesenchymal transition (EMT) process has emerged as a central regulator of embryonic development, tissue repair and tumor malignancy. In recent years, researchers have specifically focused on how mechanical signals drive the EMT program in epithelial cells. However, how epithelial cells specifically leverage mechanical force to control the EMT process remains unclear. Here, we show that the bona fide mechanically activated cation channel Piezo1 plays a critical role in the EMT. The Piezo1 is expressed in human primary epidermal keratinocytes (HEKs) and is responsible for the mechanical stretch-induced Ca2+ concentration. Inhibition of Piezo1 activation by the inhibitor GsMTx4 or by siRNA-mediated Piezo1 knockdown influenced the morphology and migration of HEKs. Moreover, Piezo1 activity also altered EMT-correlated markers expression in response to mechanical stretch. We propose that the mechanically activated cation channel Piezo1 is an important determinant of mechanical force-induced EMT in keratinocytes and might play similar roles in other epithelial cells.

Design, Physicochemical Characterization, and In Vitro Permeation of Innovative Resatorvid Topical Formulations for Targeted Skin Drug Delivery.

Nonmelanoma skin cancers (NMSCs) are the most common malignancies worldwide and affect more than 5 million people in the United States every year. NMSC is directly linked to the excessive exposure of the skin to solar ultraviolet (UV) rays. The toll-like receptor 4 (TLR4) antagonist, resatorvid (TAK-242), is a novel prototype chemo preventive agent that suppresses the production of inflammation mediators induced by UV exposure. This study aimed to design and develop TAK-242 into topical formulations using FDA-approved excipients, including DermaBaseTM, PENcreamTM, polyethylene glycol (PEG)-400, propylene glycol (PG), carbomer gel, hyaluronic acid (HA) gel, and Pluronic® F-127 poloxamer triblock copolymer gel for the prevention of skin cancer. The physicochemical properties of raw TAK-242, which influence the compatibility and solubility in the selected base materials, were confirmed using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), hot-stage microscopy (HSM), Raman spectroscopy, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopic analysis. The permeation behavior of TAK-242 from the prepared formulations was determined using Strat-M® transdermal diffusion membranes, and 3D cultured primary human-derived epidermal keratinocytes (EpiDermTM). Despite TAK-242′s high molecular weight and hydrophobicity, it can permeate through reconstructed human epidermis from all formulations. The findings, reported for the first time in this study, emphasize the capabilities of the topical application of TAK-242 via these multiple innovative topical drug delivery formulation platforms.

Nanostructured lipid carriers loaded with an association of minoxidil and latanoprost for targeted topical therapy of alopecia

Alopecia is a condition associated with different etiologies, ranging from hormonal changes to chemotherapy, that affects over 80 million people in the USA. Nevertheless, there are currently few FDA-approved drugs for topical treatment, and existing formulations still present skin irritation issues, compromising treatment adherence. This work aimed to develop a safe formulation based on nanostructured lipid carriers (NLC) that entrap an association of minoxidil and latanoprost and target drug delivery to the hair follicles. To do so, thermal techniques combined with FTIR were used to assess the chemical compatibility of the proposed drug association. Then, NLC with 393.5 ± 36.0 nm (PdI < 0.4) and +22.5 ± 0.2 mV zeta potential were produced and shown to entrap 86.9% of minoxidil and 99.9% of latanoprost efficiently. In vitro, the free drug combination was indicated to exert positive effects over human primary epidermal keratinocytes, supporting cell proliferation, migration and inducing the mRNA expression of MKI67 proliferation marker and VEGF – a possible effector for minoxidil-mediated hair growth. Interestingly, such a favorable drug combination profile was optimized when delivered using our NLC. Furthermore, according to the HET-CAM and reconstructed human epidermis assays, the nanoformulation was well tolerated. Finally, drug penetration was evaluated in vitro using porcine skin. Such experiments indicated that the NLC could be deposited preferentially into the hair follicles, causing a considerable increase in the penetration of the two drugs in such structures, compared to the control (composed of the free compounds) and generating a target-effect of approximately 50% for both drugs. In summary, present results suggest that hair follicle-targeted delivery of the minoxidil and latanoprost combination is a promising alternative to treat alopecia.

Paclitaxel-Induced Epidermal Alterations: An In Vitro Preclinical Assessment in Primary Keratinocytes and in a 3D Epidermis Model.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent approved for the treatment of ovarian, non-small cell lung, head, neck, and breast cancers. Despite its beneficial effects on cancer and widespread use, paclitaxel also damages healthy tissues, including the skin. However, the mechanisms that drive these skin adverse events are not clearly understood. In the present study, we demonstrated, by using both primary epidermal keratinocytes (NHEK) and a 3D epidermis model, that paclitaxel impairs different cellular processes: paclitaxel increased the release of IL-1α, IL-6, and IL-8 inflammatory cytokines, produced reactive oxygen species (ROS) release and apoptosis, and reduced the endothelial tube formation in the dermal microvascular endothelial cells (HDMEC). Some of the mechanisms driving these adverse skin events in vitro are mediated by the activation of toll-like receptor 4 (TLR-4), which phosphorylate transcription of nuclear factor kappa B (NF-κb). This is the first study analyzing paclitaxel effects on healthy human epidermal cells with an epidermis 3D model, and will help in understanding paclitaxel’s effects on the skin.

Phytochemical Analysis, Antioxidant, and Wound Healing Activity of Pluchea indica L. (Less) Branch Extract Nanoparticles.

Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5-O-dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 μM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

Bifidobacterium longum and Galactooligosaccharide Improve Skin Barrier Dysfunction and Atopic Dermatitis-like Skin.

PurposeThe beneficial effects of a combination therapy using Bifidobacterium longum and galactooligosaccharide (GOS) for the treatment of atopic dermatitis (AD) have not been elucidated.MethodsGene expressions of interleukin (IL)-4 and IL-13 from peripheral blood mononuclear cells and fecal abundance of B. longum from 12-month-old infants were evaluated. Human primary epidermal keratinocytes (HEKs) and hairless mice were treated with B. longum, GOS, B. longum-derived extracellular vesicles (BLEVs), dinitrochlorobenzene (DNCB), or a synbiotic mixture of B. longum and GOS. Expression of epidermal barrier proteins and cytokines as well as serum immunoglobulin E (IgE) levels were analyzed in HEKs and mice. Dermatitis scores, transepidermal water loss (TEWL), epidermal thickness, and fecal B. longum abundance were evaluated in mice.ResultsFecal abundance of B. longum was negatively correlated with blood IL-13 expression in infants. B. longum or BLEVs increased expression of filaggrin (FLG) and loricrin (LOR) in HEKs. B. longum increased the efficacy of GOS to upregulate FLG and LOR expressions in HEKs. Oral administration of GOS increased fecal abundance of B. longum in mice. Oral administration of B. longum attenuated DNCB-induced skin inflammation, abnormal TEWL, AD-like skin, and deficiency of epidermal barrier proteins. Moreover, the combination of B. longum and GOS showed greater effects to improve DNCB-induced skin inflammation, abnormal TEWL, AD-like skin, serum IgE levels, IL-4 over-expression, and the deficiency of epidermal barrier proteins than the administration of B. longum alone.ConclusionsB. longum and GOS improve DNCB-induced skin barrier dysfunction and AD-like skin.

Effect of the flavonoids quercetin and taxifolin on UVA-induced damage to human primary skin keratinocytes and fibroblasts.

The ultraviolet (UV) part of solar radiation can permanently affect skin tissue. UVA photons represent the most abundant UV component and stimulate the formation of intracellular reactive oxygen species (ROS), leading to oxidative damage to various biomolecules. Several plant-derived polyphenols are known as effective photoprotective agents. This study evaluated the potential of quercetin (QE) and its structurally related flavonoid taxifolin (TA) to reduce UVA-caused damage to human primary dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK) obtained from identical donors. Cells pre-treated with QE or TA (1h) were then exposed to UVA light using a solar simulator. Both flavonoids effectively prevented oxidative damage, such as ROS generation, glutathione depletion, single-strand breaks formation and caspase-3 activation in NHDF. These protective effects were accompanied by stimulation of Nrf2 nuclear translocation, found in non-irradiated and irradiated NHDF and NHEK, and expression of antioxidant proteins, such as heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and catalase. For most parameters, QE was more potent than TA. On the other hand, TA demonstrated protection within the whole concentration range, while QE lost its protective ability at the highest concentration tested (75μM), suggesting its pro-oxidative potential. In summary, QE and TA demonstrated UVA-protective properties in NHEK and NHDF obtained from identical donors. However, due to the in vitro phototoxic potential of QE, published elsewhere and discussed herein, further studies are needed to evaluate QE safety in dermatological application for humans as well as to confirm our results on human skin ex vivo and in clinical trials.