Highly stable and fast-dissolving ascorbic acid-loaded microneedles.

OBJECTIVES: Ascorbic acid has many benefits to the skin. Numerous attempts to promote its topical delivery show great challenges since its chemical instability and poor skin impermeability. Microneedle delivery is a simple, safe, painless and effective means to deliver therapeutic or nourishing molecules into the skin. The purpose of this study was twofold: (a) to develop a new formulation of ascorbic acid-loaded microneedles to enhance ascorbic acid stability by investigating an optimal amount of polyethyleneimine as an additive to the dextran-based microneedle formulation and (b) to assess microneedle properties in terms of dissolving rate, skin penetration ability, biocompatibility and antimicrobial activity. METHODS: The microneedles formulated with ascorbic acid and varied polyethyleneimine concentrations were fabricated and subsequently tested for ascorbic acid stability using 2,2-diphenyl-1-picrylhydrazyl assay. The dissolution rate and skin penetration depth were investigated in porcine skin and the reconstructed human full-thickness skin model respectively. The skin irritation tests were done according to the Organisation for Economic Co-operation and Development Test Guideline No. 439. An antimicrobial disc susceptibility test was performed against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. RESULTS: Among varied amounts of 0%, 1.5%, 3.0% and 4.5% (w/v), the 3.0% polyethyleneimine showed the most desirable characteristics, including well-preserved shape integrity after demoulding, significantly improved stability of ascorbic acid (p < 0.001) from 33% to 96% antioxidant activity after 8 weeks of storage at 40°C, increased dissolving rate (p < 0.001) by being completely dissolved within 2 min after the skin insertion, passing skin penetration and biocompatibility tests as well as having a broad spectrum of antimicrobial property. CONCLUSION: With a safety profile and enhanced properties, the new formulation of ascorbic acid-loaded microneedles shows outstanding potential as commercially available cosmetics and healthcare products.

OBJECTIFS: L'acide ascorbique présente de nombreux avantages pour la peau. De nombreuses tentatives pour promouvoir sa délivrance topique présentent de grands défis en raison de son instabilité chimique et de sa faible imperméabilité cutanée. L'administration de micro-aiguilles est un moyen simple, sûr, indolore et efficace d'administrer des molécules thérapeutiques ou nourrissantes dans la peau. Le but de cette étude était double : a) développer une nouvelle formulation de microaiguilles chargées d'acide ascorbique pour améliorer la stabilité de l'acide ascorbique en étudiant une quantité optimale de polyéthylèneimine comme additif à la formulation de microaiguilles à base de dextrane ; et b) évaluer les propriétés des micro-aiguilles en termes de vitesse de dissolution, de capacité de pénétration cutanée, de biocompatibilité et d'activité antimicrobienne. MÉTHODES: Les microaiguilles formulées avec de l'acide ascorbique et des concentrations variées de polyéthylèneimine ont été fabriquées et ensuite testées pour la stabilité de l'acide ascorbique à l'aide d'un dosage de 2,2-diphényl-1-picrylhydrazyle. Le taux de dissolution et la profondeur de pénétration de la peau ont été étudiés dans la peau de porc et le modèle de peau humaine reconstruite de pleine épaisseur, respectivement. Les tests d'irritation cutanée ont été effectués conformément à la ligne directrice n° 439 de l'Organisation de coopération et de développement économiques. Un test de sensibilité du disque antimicrobien a été réalisé contre Escherichia coli, Staphylococcus aureus et Staphylococcusepidermidis. RÉSULTATS: Parmi des quantités variées de 0, 1,5, 3,0 et 4,5 % (p/v), la polyéthylèneimine à 3,0 % a montré les caractéristiques les plus souhaitables, notamment une intégrité de forme bien préservée après démoulage, une stabilité significativement améliorée de l'acide ascorbique (p ⟨ 0,001) de 33 % à 96 % d'activité antioxydante après 8 semaines de stockage à 40 °C, augmentation du taux de dissolution (p ⟨ 0,001) en étant complètement dissous dans les2 minutes suivant l'insertion de la peau, en passant les tests de pénétration cutanée et de biocompatibilité, ainsi qu'en ayant un large spectre de propriétés antimicrobiennes. CONCLUSION: Avec un profil d'innocuité et des propriétés améliorées, la nouvelle formulation de micro-aiguilles chargées d'acide ascorbique présente un potentiel exceptionnel en tant que produits cosmétiques et de soins de santé disponibles dans le commerce.

Inhibition of histone deacetylase 6 destabilizes ERK phosphorylation and suppresses cancer proliferation via modulation of the tubulin acetylation-GRP78 interaction.

BACKGROUND: The leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown. METHODS: The clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT-PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells. RESULTS: HDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth. CONCLUSIONS: HDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Validation study for in vitro skin irritation test using reconstructed human skin equivalents constructed by layer-by-layer cell coating technology.

The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.

Specific Interaction of DARPin with HIV-1 CANTD Disturbs the Distribution of Gag, RNA Packaging, and Tetraspanin Remodelling in the Membrane.

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.

Tiered approach for evaluation of anti-melanogenic activity of trans-N-coumaroyltyramine derivatives.

Skin hyperpigmentation is commonly treated by topical drug application. Several naturally occurring compounds exhibit attractive biological effects including anti-melanogenic activity. Chemically modified derivatives of those compounds are expected to be more efficient. However, efficacy and safety testing processes are of significant consideration to identify the most effective compound among them. Herein, we demonstrated a tiered approach to investigate the antipigmentation activity of 17 trans-N-coumaroyltyramine derivatives. First, we evaluated the in chemico antityrosinase activity, then the cytotoxicity of the most potent derivatives using a mitochondrial activity-based assay, followed with the in vitro anti-melanogenic activity in two dimensional (2D) monolayer human melanocytes. The selected derivatives were topically applied on a three dimensional (3D) pigmented-reconstructed human epidermis (pRhE) containing melanocytes and keratinocytes to evaluate their depigmenting activity. Two of the 17 derivatives displayed a significant reduction in pigmentation in the 3D pRhE, comparable to kojic acid, a known tyrosinase inhibitor. In addition, a molecular docking experiment indicated an interaction of the three derivatives and tyrosinase, suggesting that these derivatives have potent anti-melanogenic activity through tyrosinase inhibition. Our findings provide an alternative approach for investigating skin-whitening agents, thereby facilitating the research and development of skin-whitening products that need not be tested on animals.

CAMSAP3 depletion induces lung cancer cell senescence-associated phenotypes through extracellular signal-regulated kinase inactivation.

BACKGROUND: Cellular senescence is an aging-related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a minus-end microtubule-stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood. METHODS: The function of CAMSAP3 on the regulation of cellular senescence-associated phenotypes in human non-small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence-associated phenotypes were observed by SA-ß-Gal staining. Quantitative RT-PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3-ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed. RESULTS: CAMSAP3 knockout promotes lung cancer cell senescence-associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p-ERK) was markedly downregulated in CAMSAP3-deleted cells, suppressing cyclin D1 expression levels, and full-length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3-modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p-ERK and cyclin D1, resulting in a senescence-like phenotype. CONCLUSION: This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence-associated phenotypes via the modulation of p-ERK/cyclin D1 signaling.

Neuronal growth and synaptogenesis are inhibited by prenatal methamphetamine exposure leading to memory impairment in adolescent and adult mice.

Synaptogenesis plays critical roles in learning and memory processes and is susceptible to substance abuse toxicity. The present study aimed to elucidate the long-lasting effects of prenatal methamphetamine exposure on synaptogenesis and learning and memory. The involvement of BDNF-TrkB signaling was also investigated. Pregnant mice (C57BL/6 JNc) were administered methamphetamine (5 mg/kg, s.c.) on gestation days 8-15. Primary hippocampal cultures were prepared from fetuses at gestational day 16.5 to study neuronal morphology and synaptogenesis. The expression of synaptic proteins, BDNF and TrkB receptor was determined in postnatal day 14 (PND14), adolescent and adult mice; memory tests were also conducted. MA exposure decreased axon length and diameter, and synaptic areas in the primary cultures. Presynaptic protein was decreased in the hippocampus of PND14 mice prenatally exposed to MA, while increases in postsynaptic protein (PSD-95) were found in MA-exposed adolescent and adult mice. BDNF expression was enhanced in the prefrontal cortex and striatum of MA-exposed PND14 mice. Memory impairment was observed in MA-exposed adolescent and adult mice compared to control mice. Prenatal MA exposure disrupted neuronal growth and synapse formation in the developing brain with only short-term interference of the BDNF-TrkB signaling pathway, resulting in the adaptation of postsynaptic neurons. Alterations in the developing brain and synaptogenesis lead to long-lasting learning and memory impairment.

Multilayered fibroblasts constructed by accelerated cellular self-assembly and applications for regenerative medicine.

Regenerative medicine research requires animal experiments to evaluate the treatment effects. According to the 3Rs principles, alternative models have been developed and utilized to evaluate the efficacy and safety of new products. Three-dimensional (3D) cell cultures have been recognized for their relevant structures and biological functions akin to native tissues. They can better represent in vivo conditions than two-dimensional (2D) cell cultures. Herein, we present a fast and simple technique for the construction of 3D dermal fibroblasts (3D-DFs) without exogenous scaffolds. The 3D-DFs can be obtained within 3 days by seeding DFs at a level that exceeds their confluent density and culturing in the presence of ascorbic acid. The 3D-DFs had a compact multilayer structure, as revealed from their histology. The collagen content of the resulting 3D-DFs drastically increased compared to in a monolayer. The 3D-DF-derived extracellular matrix can serve for the 3D culturing of other cells. A gap closure assay was performed with the 3D-DFs to represent a 3D-wounded dermal model. Interestingly, the multilayered structure of the 3D-DFs could be regenerated after wounding even when cultured in the absence of ascorbic acid. Moreover, skin grafting of the 3D-DFs was demonstrated in vitro using wounded full-thickness skin models as an alternative to animal experiments. The 3D-DFs will potentially be useful for regenerative medicine or as tissue models for in vitro studies.

Loss of CAMSAP3 promotes EMT via the modification of microtubule-Akt machinery.

Epithelial-to-mesenchymal transition (EMT) plays pivotal roles in a variety of biological processes, including cancer invasion. Although EMT involves alterations of cytoskeletal proteins such as microtubules, the role of microtubules in EMT is not fully understood. Microtubule dynamics are regulated by microtubule-binding proteins, and one such protein is CAMSAP3, which binds the minus-end of microtubules. Here, we show that CAMSAP3 is important to preserve the epithelial phenotypes in lung carcinoma cells. Deletion of CAMSAP3 in human lung carcinoma-derived cell lines showed that CAMSAP3-deficient cells acquired increased mesenchymal features, mostly at the transcriptional level. Analysis of the mechanisms underlying these changes demonstrated that tubulin acetylation was dramatically increased following CAMSAP3 removal, leading to the upregulation of Akt proteins (also known as protein kinase B proteins, hereafter Akt) activity, which is known to promote EMT. These findings suggest that CAMSAP3 functions to protect lung carcinoma cells against EMT by suppressing Akt activity via microtubule regulation and that CAMSAP3 loss promotes EMT in these cells.This article has an associated First Person interview with the first author of the paper.

Shape and surface properties of titanate nanomaterials influence differential cellular uptake behavior and biological responses in THP-1 cells.

We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, were synthesized from anatase TiO2 nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (∼2 µm in length) was amorphous with an acidic surface, while TF-2 (∼7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.

Use of Three-Dimensional Arterial Models To Predict the In Vivo Behavior of Nanoparticles for Drug Delivery.

Nanomaterials have been widely used for applications in biomedical fields and could become indispensable in the near future. However, since it is difficult to optimize in vivo biological behavior in a 3D environment by using a single cell in vitro, there have been many failures in animal models. In vitro prediction systems using 3D human-tissue models reflecting the 3D location of cell types may be useful to better understand the biological characteristics of nanomaterials for optimization of their function. Herein we demonstrate the potential ability of 3D engineered human-arterial models for in vitro prediction of the in vivo behavior of nanoparticles for drug delivery. These models enabled optimization of the composition and size of the nanoparticles for targeting and treatment efficacy for atherosclerosis. In vivo experiments with atherosclerotic mice suggested excellent biological characteristics and potential treatment effects of the nanoparticles optimized in vitro.

Three-dimensional human arterial wall models for in vitro permeability assessment of drug and nanocarriers.

Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular walls which have a 3D-structure and are composed of not only ECs, but also smooth muscle cells (SMCs) and other surrounding tissues. Here in, 5-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirmed by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with the known phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWMs over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(γ-Glutamic Acid) nanoparticles (γ-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000 k (28 nm), the γ-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWMs, suggesting the effect of intrinsic properties of the substance on the transport. This in vitro evaluation system using the 3D-AWMs is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis.

Survival and structural evaluations of three-dimensional tissues fabricated by the hierarchical cell manipulation technique.

Mouse L929 fibroblasts and normal human dermal fibroblasts (NHDFs) were constructed into three-dimensional (3-D) multilayered tissues by directly coating them with nano-films consisting of fibronectin (FN) and gelatin (G) onto the surface of the cell layer using layer-by-layer assembly. The one-, two- and five-layered (1L, 2L and 5L) tissues were cultured for 1 month in order to evaluate their long-term survival and structural changes. L929 cells in the 3-D tissues showed excessive proliferation throughout the culture period, regardless of the starting layer number. The cross-sectional images stained with hematoxylin and eosin revealed heterogeneous and random increases in the thickness of their layered structures, probably due to the immortalized property of L929 fibroblasts. On the other hand, NHDFs, which are primary cells, showed high stability in their amount of DNA in the multilayered structures, and their thicknesses were completely maintained even after 1 month of incubation. To evaluate the living cell density in each layer of 5L tissues during the culture period, 5L NHDFs were fluorescently labeled with LIVE/DEAD reagent and analyzed by confocal laser scanning microscopy. Although the upper layers did not show any dead cells, the bottom layers showed pieces of dead cell nuclei depending on culture time. However, the living cell densities in all layers were almost the same, even after 1 month of culture, suggesting that the 5L structures were completely filled with living cells. These findings from the multilayered tissue constructs will provide important information not only for the construction of 3-D engineered tissues in tissue engineering but also on 3-D cell culture in biological science generally.