Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid.

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca²âº in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.

Dental optical coherence tomography: new potential diagnostic system for cracked-tooth syndrome.

PURPOSE: The aim of the present study was to determine the reliability of optical coherence tomography (OCT) in detecting cracked teeth and its relative clinical effectiveness by comparing it with other diagnostic methods including conventional visual inspection, trans-illumination, and micro-computed tomography (micro-CT). METHODS: The reliability of swept source OCT (SS-OCT) was verified by comparing the number of detected crack lines on 109 surfaces of 61 teeth with those detected with other conventional methods. RESULTS: One to one comparison revealed that crack lines that were invisible with naked eyes could be found in SS-OCT images. The detection ability of SS-OCT was superior or similar to those of micro-CT (100.0 %) and trans-illumination. CONCLUSIONS: Crack lines shown in the SS-OCT images had distinct characteristics, and structural crack lines and craze lines could be distinguished in SS-OCT images. Thus, the detection ability of SS-OCT renders it an acceptable diagnostic device for cracked-tooth syndrome.

A novel near-infrared fluorescence chemosensor for copper ion detection using click ligation and energy transfer.

A novel near-infrared fluorescence (NIRF) copper sensor allows rapid and ultra-sensitive detection of copper ions with excellent selectivity and specificity due to the specificity of click ligation and effective dark-quenching mechanism.

Robust mapping of the myelin water fraction in the presence of noise: synergic combination of anisotropic diffusion filter and spatially regularized nonnegative least squares algorithm.

PURPOSE: To improve the mapping of myelin water fraction (MWF) despite the presence of measurement noise, and to increase the visibility of fine structures in MWF maps. MATERIALS AND METHODS: An anisotropic diffusion filter (ADF) was effectively combined with a spatially regularized nonnegative least squares algorithm (srNNLS) for robust MWF mapping. Synthetic data simulations were performed to assess the effectiveness of this new method. Experimental measurements of signal decay curves were obtained and MWF maps were estimated using the new method and compared with maps estimated using other methods. RESULTS: MWF mapping was substantially improved in both simulations and experimental data when ADF was combined with the srNNLS algorithm. MWF variability decreased with the use of the proposed method, which in turn resulted in increased visibility of small focal lesions and structures in the MWF maps. CONCLUSION: This study demonstrates that the benefits of ADF and srNNLS algorithms can be effectively combined in a synergic way for robust mapping of MWF in the presence of noise. Substantial improvements to MWF mapping can be made using the proposed method.

Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe.

We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.

Cellular uptake pathway and drug release characteristics of drug-encapsulated glycol chitosan nanoparticles in live cells.

Herein, we evaluated the cellular uptake pathways of hydrophobically modified glycol chitosan (HGC) nanoparticles as nano-sized drug carriers using cellular imaging technology. The endocytic pathway of nanocarriers for intracellular drug delivery is of great interest for the design of high efficacy delivery carriers for therapeutic agents. To evaluate the cellular uptake pathways of HGC nanoparticles, HGC was chemically labeled with near infrared (NIR) fluorescence dye, Cy5.5, to visualize the nanoparticle under confocal laser scanning microscopy. The internalization pathways of HGC nanoparticles were evaluated after treatment of specific endocytosis inhibitors. Importantly, HCG nanoparticles showed different cellular uptake efficiency and intracellular fate in cytoplasm according to the internalization pathways. Furthermore, drug distribution also evaluated according to the endocytic pathways after treatment of drug encapsulated HGC nanoparticles. As a model drug, fluorescent photosensitizer, Ce6, was encapsulated into HGC (Ce6-HGC) nanoparticles and the distribution of Ce6 in cytoplasm was evaluated using confocal laser scanning microscopy. The intracellular drug distribution showed different manner through specific endocytic pathways. The cellular imaging technology is highly useful for evaluation of endocytosis pathways and intracellular fate of drug delivery carrier which are closely related to drug distribution and therapeutic efficacy.

Tumor-homing glycol chitosan/polyethylenimine nanoparticles for the systemic delivery of siRNA in tumor-bearing mice.

Here, we designed a new nano-sized siRNA carrier system composed of biocompatible/biodegradable glycol chitosan polymer (GC) and strongly positively charged polyethylenimine (PEI) polymers. In order to make a stable and tumor-homing nano-sized carrier, each polymer was modified with hydrophobic 5beta-cholanic acid, and they were simply mixed to form self-assembled GC-PEI nanoparticles (GC-PEI NPs), due to the strong hydrophobic interactions of 5beta-cholanic acids in the polymers. The freshly prepared GC-PEI NPs showed a stable nanoparticle structure (350nm) and they presented a strongly positive-charged surface (zeta potential=23.8) that is enough to complex tightly with negatively charged RFP-siRNAs, designed for inhibiting red fluorescent protein (RFP) expression. The siRNA encapsulated nanoparticles (siRNA-GC-PEI NPs) formed more compact and stable nanoparticle structures (250nm) at 1: 5 weight ratio of siRNA to GC-PEI nanoparticles. In vitro RFP expressing B16F10 tumor cell (RFP/B16F10) culture system, the siRNA-GC-PEI NPs presented a rapid time-dependent cellular uptake profile within 1h. Moreover, the internalized siRNA-GC-PEI NPs lead to specific mRNA breaks down. Furthermore, our new formulation of siRNA-GC-PEI NPs presented a significant inhibition of RFP gene expression of RFP/B16F10-bearing mice, due to their higher tumor-targeting ability. These results revealed the promising potential of GC-PEI NPs as a stable and effective nano-sized siRNA delivery system for cancer treatment.

Self-assembled hyaluronic acid nanoparticles for active tumor targeting.

Hyaluronic acid nanoparticles (HA-NPs), which are formed by the self-assembly of hydrophobically modified HA derivatives, were prepared to investigate their physicochemical characteristics and fates in tumor-bearing mice after systemic administration. The particle sizes of HA-NPs were controlled in the range of 237-424 nm by varying the degree of substitution of the hydrophobic moiety. When SCC7 cancer cells over-expressing CD44 (the receptor for HA) were treated with fluorescently labeled Cy5.5-HA-NPs, strong fluorescence signals were observed in the cytosol of these cells, suggesting efficient intracellular uptake of HA-NPs by receptor-mediated endocytosis. In contrast, no significant fluorescence signals were observed when Cy5.5-labeled HA-NPs were incubated with normal fibroblast cells (CV-1) or with excess free-HA treated SCC7 cells. Following systemic administration of Cy5.5-labeled HA-NPs with different particle sizes into a tumor-bearing mouse, their biodistribution was monitored as a function of time using a non-invasive near-infrared fluorescence imaging system. Irrespective of the particle size, significant amounts of HA-NPs circulated for two days in the bloodstream and were selectively accumulated into the tumor site. The smaller HA-NPs were able to reach the tumor site more effectively than larger HA-NPs. Interestingly, the concentration of HA-NPs in the tumor site was dramatically reduced when mice were pretreated with an excess of free-HA. These results imply that HA-NPs can accumulate into the tumor site by a combination of passive and active targeting mechanisms.

Stability and cellular uptake of polymerized siRNA (poly-siRNA)/polyethylenimine (PEI) complexes for efficient gene silencing.

Small interfering RNA (siRNA) is a promising biological strategy for treatment of diverse diseases, but the therapeutic application of siRNA has been limited by its instability and poor cellular uptake efficiency. Although the development of various gene delivery systems has increased the siRNA delivery efficiency, many problems still remain to be resolved before the clinical application of siRNA. In this study, we suggest reducible polymerized siRNA a possible solution for low delivery efficiency of siRNA. Dithiol-modified red fluorescent protein (RFP) siRNAs at the 5'-ends of both sense and anti-sense strands were disulfide-polymerized. Polymerized siRNA (poly-siRNA) was composed of 30% oligomeric siRNA (50 approximately 300 bps) and 66% polymeric siRNA (above approximately 300 bps) as fractions, and was reducible in reducing solution through disulfide bond cleavage. Poly-siRNA formed more condensed and nano-sized complexes with low molecular weight polyethylenimine (PEI) by strong electrostatic interaction based on the higher charge density of poly-siRNA, compared with siRNA (mono-siRNA). The compact poly-siRNA/PEI complexes prevented the loss and degradation of siRNA from a polyanion competitor and RNases in serum. Furthermore, poly-siRNA/PEI complexes exhibited superior intracellular uptake by murine melanoma cells (B16F10), and was accompanied with RFP gene silencing efficiency of about 80%, compared to untreated cells. These results sufficiently support that strong polyanionic and reducible poly-siRNA can be utilized as a novel powerful therapeutic strategy for human diseases.

Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles.

Polymeric nanoparticle-based carriers are promising agents for the targeted delivery of therapeutics to the intracellular site of action. To optimize the efficacy in delivery, often the tuning of physicochemical properties (i.e., particle size, shape, surface charge, lipophilicity, etc.) is necessary, in a manner specific to each type of nanoparticle. Recent studies showed an efficient tumor targeting by hydrophobically modified glycol chitosan (HGC) nanoparticles through the enhanced permeability and retention (EPR) effect. As a continued effort, here the investigations on the cellular uptake mechanism and the intracellular fate of the HGC nanoparticles are reported. The HGC nanoparticle, prepared by a partial derivatization of the free amino groups of glycol chitosan (GC) with 5beta-cholanic acid, had a globular shape with the average diameter of 359 nm and the zeta potential of ca. 22 mV. Interestingly, these nanoparticles showed an enhanced distribution in the whole cells, compared to the parent hydrophilic GC polymers. In vitro experiments with endocytic inhibitors suggested that several distinct uptake pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) are involved in the internalization of HGC. Some HGC nanoparticles were found entrapped in the lysosomes upon entry, as determined by TEM and colocalization studies. Given such favorable properties including low toxicity, biocompatibility, and fast uptake by several nondestructive endocytic pathways, our HGC nanoparticles may serve as a versatile carrier for the intracellular delivery of therapeutic agents.