Rapid Communication The Phototumorigenic Fluoroquinolone Lomefloxacin Photosensitizes Pyrimidine Dimer Formation in Human Keratinocytes In Vitro

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

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Cryptotanshinone from Salvia miltiorrhiza Bunge lowers the Cytokeratin 1/10 expression in primary human Keratinocytes in vitro

10.1055/s-0037-1608052 ◽

2017 ◽

Author(s):

S Esch ◽

A Hensel ◽

S Brandt ◽

S König

Keyword(s):

Salvia Miltiorrhiza ◽

Human Keratinocytes ◽

Salvia Miltiorrhiza Bunge ◽

Primary Human Keratinocytes

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Dexamethasone-Loaded Lipomers: Development, Characterization, and Skin Biodistribution Studies

Pharmaceutics ◽

10.3390/pharmaceutics13040533 ◽

2021 ◽

Vol 13 (4) ◽

pp. 533

Author(s):

Eloy Pena-Rodríguez ◽

Maria Lajarin-Reinares ◽

Aida Mata-Ventosa ◽

Sandra Pérez-Torras ◽

Francisco Fernández-Campos

Keyword(s):

In Vitro Release ◽

Human Keratinocytes ◽

Topical Administration ◽

In Vitro Cytotoxicity ◽

Hair Follicles ◽

Vertical Diffusion ◽

Biodistribution Studies ◽

Follicular Targeting ◽

Depot Effect

Follicular targeting has gained more attention in recent decades, due to the possibility of obtaining a depot effect in topical administration and its potential as a tool to treat hair follicle-related diseases. Lipid core ethyl cellulose lipomers were developed and optimized, following which characterization of their physicochemical properties was carried out. Dexamethasone was encapsulated in the lipomers (size, 115 nm; polydispersity, 0.24; zeta-potential (Z-potential), +30 mV) and their in vitro release profiles against dexamethasone in solution were investigated by vertical diffusion Franz cells. The skin biodistribution of the fluorescent-loaded lipomers was observed using confocal microscopy, demonstrating the accumulation of both lipomers and fluorochromes in the hair follicles of pig skin. To confirm this fact, immunofluorescence of the dexamethasone-loaded lipomers was carried out in pig hair follicles. The anti-inflammatory (via TNFα) efficacy of the dexamethasone-loaded lipomers was demonstrated in vitro in an HEK001 human keratinocytes cell culture and the in vitro cytotoxicity of the nanoformulation was investigated.

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Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽

10.3390/biom11060915 ◽

2021 ◽

Vol 11 (6) ◽

pp. 915

Author(s):

Jazelli Mueterthies ◽

Davit A. Potoyan

Keyword(s):

Phase Separation ◽

Low Complexity ◽

Nuclear Bodies ◽

Disordered Proteins ◽

Solvent Exposure ◽

Ion Release ◽

Dimer Formation ◽

Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

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Ethosomes and Transethosomes for Mangiferin Transdermal Delivery

Antioxidants ◽

10.3390/antiox10050768 ◽

2021 ◽

Vol 10 (5) ◽

pp. 768

Author(s):

Maddalena Sguizzato ◽

Francesca Ferrara ◽

Supandeep Singh Hallan ◽

Anna Baldisserotto ◽

Markus Drechsler ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Radical Scavenging ◽

Human Keratinocytes ◽

Antioxidant Response ◽

Correlation Spectroscopy ◽

Inflammatory Status ◽

Transmission Electron ◽

Anti Inflammatory

Mangiferin is a natural glucosyl xanthone with antioxidant and anti-inflammatory activity, making it suitable for protection against cutaneous diseases. In this study ethosomes and transethosomes were designed as topical delivery systems for mangiferin. A preformulation study was conducted using different surfactants in association with phosphatidylcholine. Vesicle dimensional distribution was monitored by photon correlation spectroscopy, while antioxidant capacity and cytotoxicity were respectively assessed by free radical scavenging analysis and MTT on HaCaT keratinocytes. Selected nanosystems were further investigated by cryogenic transmission electron microscopy, while mangiferin entrapment capacity was evaluated by ultracentrifugation and HPLC. The diffusion kinetics of mangiferin from ethosomes and transethosomes evaluated by Franz cell was faster in the case of transethosomes. The suitability of mangiferin-containing nanovesicles in the treatment of skin disorders related to pollutants was investigated, evaluating, in vitro, the antioxidant and anti-inflammatory effect of ethosomes and transethosomes on human keratinocytes exposed to cigarette smoke as an oxidative and inflammatory challenger. The ability to induce an antioxidant response (HO-1) and anti-inflammatory status (IL-6 and NF-kB) was determined by RT-PCR and immunofluorescence. The data demonstrated the effectiveness of mangiferin loaded in nanosystems to protect cells from damage. Finally, to gain insight into the keratinocytes’ uptake of ethosome and transethosome, transmission electron microscopy analyses were conducted, showing that both nanosystems were able to pass intact within the cells.

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Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

Molecules ◽

10.3390/molecules26082153 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2153

Author(s):

Raffaella Marina Lecci ◽

Isabella D’Antuono ◽

Angela Cardinali ◽

Antonella Garbetta ◽

Vito Linsalata ◽

...

Keyword(s):

Antioxidant Activities ◽

Biological Activities ◽

Human Keratinocytes ◽

Ldl Oxidation ◽

Trolox Equivalent Antioxidant Capacity ◽

Epidermal Keratinocytes ◽

Olive Cultivar ◽

Human Epidermal Keratinocytes ◽

Apoptotic Effect

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.

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Positively Charged Lipid as Potential Tool to Influence the Fate of Ethosomes

Applied Sciences ◽

10.3390/app11157060 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7060

Author(s):

Antonia Mancuso ◽

Maria Chiara Cristiano ◽

Massimo Fresta ◽

Daniele Torella ◽

Donatella Paolino

Keyword(s):

Cell Model ◽

Human Keratinocytes ◽

Skin Delivery ◽

Physico Chemical ◽

Cell Mortality ◽

Mean Size ◽

A Cell ◽

Negative Surface ◽

Cytotoxic Studies

Ethosomes® are one of the main deformable vesicles proposed to overcome the stratum corneum. They are composed of lecithin, ethanol and water, resulting in round vesicles characterized by a narrow size distribution and a negative surface charge. Taking into account their efficiency to deliver drugs into deeper skin layers, the current study was designed to evaluate the influence of different lipids on the physico-chemical features of traditional ethosomes in the attempt to influence their fate. Three lipids (DOPE, DSPE and DOTAP) were used for the study, but only DOTAP conferred a net positive charge to ethosomes, maintaining a narrow mean size lower than 300 nm and a good polydispersity index. Stability and in vitro cytotoxic studies have been performed using Turbiscan Lab analysis and MTT dye exclusion assay, respectively. Data recorded demonstrated the good stability of modified ethosomes and a reasonable absence of cell mortality when applied to human keratinocytes, NCTC 2544, which are used as a cell model. Finally, the best formulations were selected to evaluate their ability to encapsulate drugs, through the use of model compounds. Cationic ethosomes encapsulated oil red o and rhodamine b in amounts comparable to those recorded from conventional ethosomes (over 50%). Results recorded from this study are encouraging as cationic ethosomes may open new opportunities for skin delivery.

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Keratinocytes Regulate the Threshold of Inflammation by Inhibiting T Cell Effector Functions

Cells ◽

10.3390/cells10071606 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1606

Author(s):

Peter Seiringer ◽

Stefanie Eyerich ◽

Kilian Eyerich ◽

Daniela Dittlein ◽

Anna Caroline Pilz ◽

...

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Cytokine Production ◽

Nickel Sulfate ◽

Human Keratinocytes ◽

T Cell Proliferation ◽

Effector Functions ◽

The Impact ◽

Cell Effector

Whilst the importance of keratinocytes as a first-line defense has been widely investigated, little is known about their interactions with non-resident immune cells. In this study, the impact of human keratinocytes on T cell effector functions was analyzed in an antigen-specific in vitro model of allergic contact dermatitis (ACD) to nickel sulfate. Keratinocytes partially inhibited T cell proliferation and cytokine production. This effect was dependent on the keratinocyte/T cell ratio and was partially reversible by increasing the number of autologous dendritic cells. The inhibition of T cell proliferation by keratinocytes was independent of the T cell subtype and antigen presentation by different professional antigen-presenting cells. Autologous and heterologous keratinocytes showed comparable effects, while the fixation of keratinocytes with paraformaldehyde abrogated the immunosuppressive effect. The separation of keratinocytes and T cells by a transwell chamber, as well as a cell-free keratinocyte supernatant, inhibited T cell effector functions to the same amount as directly co-cultured keratinocytes, thus proving that soluble factor/s account for the observed suppressive effects. In conclusion, keratinocytes critically control the threshold of inflammatory processes in the skin by inhibiting T cell proliferation and cytokine production.

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