Sangelose-based gels and films: effects of glycerol and α-cyclodextrin and their pharmaceutical application.

OBJECTIVE: To evaluate the possible application of Sangelose as an alternative to gelatin and carrageenan for the development of film substrates, and to examine the effect of glycerol and α-cyclodextrin (α-CyD) on the viscoelastic properties of Sangelose-based gels and the physical properties of the films. SIGNIFICANCE: Sangelose-based gels/films can serve as a potential viable alternative to gelatin and carrageenan in pharmaceutical applications. METHODS: Glycerol (a plasticizer) and α-CyD (a functional additive) were added to Sangelose, and gels and films were prepared. The gels were evaluated by dynamic viscoelasticity measurements, and the films were evaluated by scanning electron microscopy, Fourier-transform infrared spectroscopy, tensile tests, and contact angle measurements. Soft capsules were prepared using the formulated gels. RESULTS: The strength of the gels was affected when only glycerol was added to Sangelose and α-CyD addition resulted in rigid gels. However, the addition of α-CyD with 10% glycerol weakened the gels. Tensile tests suggested that glycerol addition affected the formability and malleability of the films, while α-CyD addition affected their formability and elongation properties. The addition of 10% glycerol and α-CyD did not affect the flexibility of the films, suggesting that the malleability and strength were impacted. Soft capsules could not be prepared by adding only glycerol or α-CyD to Sangelose. Soft capsules with favorable disintegration behavior were obtained upon adding α-CyD to gels along with 10% glycerol. CONCLUSIONS: Sangelose combined with a suitable amount of glycerol and α-CyD has preferable characteristics for film formation and may have potential applications in the pharmaceutical and health food sectors.

Impact of Properties of Hydrated Silicon Dioxide as Core Material on the Characteristics of Drug-containing Particles Prepared by the 2-step Process Melt Granulation Technology, MALCORE®.

Drug-containing particles (DCPs) are frequently used as cores in the development of solid oral dosage forms. The wet layering technique, which is a typical approach for preparing DCPs, requires the use of solvents and a long manufacturing time. In our previous study, we developed a novel manufacturing technology, MALCORE®, which can solve these problems through melt granulation. However, particle size control methods for DCPs in MALCORE® and the effect of the physical properties of the hydrated silicon dioxide (HSD) used for the core have not been clarified. The aim of this study was to examine the effects of the particle and pore sizes of HSD on the properties of the prepared DCPs. The results showed that the DCPs prepared using MALCORE® could be controlled by the particle size of HSD. The drug-loading efficiency tended to decrease as HSD particle size increased. Additionally, the amount of drug layering in DCPs increased as the pore size of HSD increased, but HSDs with a pore size much larger than the particle size were not able to properly layer the drug. These findings are helpful for applying MALCORE® to a variety of oral drug formulations.

A New Approach for Characterizing the Thixotropic Properties of Gel Formulations as Sprayable Agents Based on Rheological Analysis.

The present study evaluated the rheological properties of gel formulations composed of the thixotropic peptide amphiphile, palmitoyl-glycine-histidine (Pal-GH), and the thickener, propylene glycol alginate (PGA), to propose a proper approach to design sprayable gel formulations with good spray performance and high retention of a therapeutic agent. The hysteresis loop area (HLA), a conventional index of thixotropy, was determined from the relationship between the shear stress and shear rate of various formulations with different amounts of Pal-GH and PGA. In addition, a new assessment method for characterizing the thixotropy using the initial structure recovery speed was determined based on the time course of the complex modulus (G*) after structural breakdown of the gel formulations. The G* values increased with the increase in the amount of Pal-GH and PGA, indicating that the formulations were not deformable. Additionally, high HLA and high initial structure recovery speed are preferable when selecting a formulation with good spray performance and high retention. As suitable combinations of Pal-GH and PGA could exhibit both high HLA and high initial structure recovery speed, they are promising components for gel formulations to be used as sprayable agents with good spray performance and high retention. The results also suggested that the initial structure recovery speed would reflect the thixotropy for the formulation more appropriately than HLA. Thus, the initial structure recovery speed could be a useful scale for the preparation of sprayable gel formulations.

Development of a Liquid Crystal Formulation that Can Penetrate the Stratum Corneum for Intradermal Delivery of Small Interfering RNA.

Topical delivery of small interfering RNA (siRNA) can be an attractive method for the treatment of skin diseases and improving the quality of life of patients. However, it is difficult for siRNA to pass through the two major barriers of the skin: the stratum corneum (SC) and tight junctions. We have previously reported that atopic dermatitis of skin without the SC can be efficiently treated by the intradermal administration of trans-activator of transcription (Tat) peptide and AT1002 (tight junction opening peptide). However, novel drug delivery systems are needed for effective SC penetration. Therefore, in the present study, we aimed to develop a lyotropic liquid crystalline (LC) system containing Tat and AT1002 for effective siRNA penetration through the SC. An LC formulation was prepared using selachyl alcohol and purified water, and its skin penetration ability was evaluated. No fluorescence was observed in mouse skin treated with a siRNA solution, as there was no intradermal localization of siRNA from naked siRNA. However, intradermal delivery of siRNA was remarkable and extensive with the LC formulation containing both Tat and AT1002. Semiquantitative analysis by brightness measurement revealed that the LC formulation containing both Tat and AT1002 had significantly enhanced intact skin permeability than other formulations. These results show that the functional peptides in the LC formulation increased SC penetration and intradermal delivery in the healthy skin. Therefore, this novel LC system may be useful in the treatment of various skin diseases.

Electroporation-Based ex Vivo Gene Delivery into Dendritic Cells by Anionic Polymer-Coated Versatile Nuclear Localization Signal/pDNA Complex.

In this study, we focused on a nuclear localization signal (NLS)-based versatile peptide vector, designed by us, combined with electroporation (EP) to establish an efficient gene delivery system to non-dividing or slow growing dendritic cells. We determined the intranuclear transport, gene expression, and cell viability in JAWS II mouse dendritic cells transfected with the green fluorescent protein (GFP) expression plasmid DNA alone (naked pEGFP); positive charged complex of NLS derivative STR-CH2SV40H2C, and pEGFP (binary complex); or negative charged complex of the binary complex with a biocompatible polyanion, γ-polyglutamic acid (ternary complex) combined with or without EP application. Although the binary complex showed higher nuclear transport and GFP expression in the absence of EP than those for naked pEGFP, the combination of EP significantly decreased the cell viability and did not improve the efficiency of compared gene expression. However, in the ternary complex, the intranuclear transport and GFP expression efficiency were significantly higher than those of naked pEGFP and the binary complex when combined with EP, and there was no decrease in cell viability. The results suggest that polyanion-coated ternary complex with EP is useful for non-viral gene delivery system into non-dividing cells for ex vivo gene/cell therapy.

Anti-RelA siRNA-Encapsulated Flexible Liposome with Tight Junction-Opening Peptide as a Non-invasive Topical Therapeutic for Atopic Dermatitis.

Small interfering RNA (siRNA) has been proposed as a novel treatment for atopic dermatitis (AD) because it suppresses sequence-specific mRNA expression. Indeed siRNA-based therapy achieves an almost complete cure with fewer side effects than currently available treatments. However, the tight junctions in the granular layer of the epidermis in the atopic skin are barriers to siRNA delivery. We previously reported the potential clinical utility of AT1002, a peptide that opens tight junctions. In the present study, we evaluated a topical siRNA-based therapy for AD using AT1002 in combination with a flexible liposome. The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS) liposome was chosen as a carrier for siRNA because of its highly flexible structure and permeability. We prepared siRNA-encapsulated DOPE/CHEMS liposomes and examined their physical properties, safety, uptake into RAW264.7 cells, and topical application in healthy and AD-affected skin. We then assessed the efficacy of anti-nuclear factor-kappa B (NF-κB) (RelA) siRNA (siRelA)-encapsulated DOPE/CHEMS liposomes with AT1002 in AD model mice. The siRNA-DOPE/CHEMS liposomes were absorbed significantly better than siRNA alone and they enhanced siRNA skin penetration without toxicity. Moreover, siRelA-DOPE/CHEMS liposomes with AT1002 alleviated AD symptoms and reduced the levels of inflammatory cytokines in AD model mice. Therefore, the combination of AT1002 and DOPE/CHEMS liposomes could be a dermally applied RNA interference therapeutic system for effective RNA delivery and AD treatment.

Imidazo[1,2-a]pyridin-6-yl-benzamide analogs as potent RAF inhibitors.

A series of imidazo[1,2-a]pyridin-6-yl-benzamide analogs was designed as inhibitors of B-RAFV600E. Medicinal chemistry techniques were employed to explore the SAR for this series and improve selectivity versus P38 and VEGFR2.

Intra-articular Retention and Anti-arthritic Effects in Collagen-Induced Arthritis Model Mice by Injectable Small Interfering RNA Containing Hydrogel.

Small interfering RNAs (siRNAs) are expected to offer a means of treating rheumatoid arthritis (RA) because they allow the specific silencing of genes related to RA pathogenesis. In our previous study, we reported that the siRNA targeted against RelA (anti-RelA siRNA), an important nuclear factor-kappaB (NF-κB) subdomain, was an effective therapeutic in atopic dermatitis and RA model animals. In this study, to develop an intra-articular injectable gel formulation against RA, we prepared a hydrogel that contains anti-RelA siRNA, and determined the in vitro release profile (%) and in vivo intra-articular retention of fluorescence-labeled model siRNA, and the anti-arthritic effects of the anti-RelA siRelA containing hydrogel in RA model mice. We selected the silk protein, sericin (SC), as an aqueous gel base, as it is a biocompatible and useful for forming hydrogels without a cross-linker. We showed that fluorescence-labeled model siRNA was continuously released from SC hydrogel in vitro, and retained in the knee joint of rats after injection of siRNA hydrogel. In addition, the knee joint thickness, clinical severity and incidence (%) in collagen-induced arthritis (CIA) mice as RA model treated with anti-RelA siRNA containing hydrogel were more improved than untreated, anti-RelA siRNA solution and negative control siRNA containing hydrogel group. Therefore, the intra-articular injectable sericin hydrogel formulation containing of anti-RelA siRNA could be a great potential therapeutic in rheumatoid arthritis.

Development of an Innovative Intradermal siRNA Delivery System Using a Combination of a Functional Stearylated Cytoplasm-Responsive Peptide and a Tight Junction-Opening Peptide.

As a new category of therapeutics for skin diseases including atopic dermatitis (AD), nucleic acids are gaining importance in the clinical setting. Intradermal administration is noninvasive and improves patients' quality of life. However, intradermal small interfering RNA (siRNA) delivery is difficult because of two barriers encountered in the skin: intercellular lipids in the stratum corneum and tight junctions in the stratum granulosum. Tight junctions are the major barrier in AD; therefore, we focused on functional peptides to devise an intradermal siRNA delivery system for topical skin application. In this study, we examined intradermal siRNA permeability in the tape-stripped (20 times) back skin of mice or AD-like skin of auricles treated with 6-carboxyfluorescein-aminohexyl phosphoramidite (FAM)-labeled siRNA, the tight junction modulator AT1002, and the functional cytoplasm-responsive stearylated peptide STR-CH2R4H2C by using confocal laser microscopy. We found that strong fluorescence was observed deep and wide in the epidermis and dermis of back skin and AD-like ears after siRNA with STR-CH2R4H2C and AT1002 treatment. After 10 h from administration, brightness of FAM-siRNA was significantly higher for STR-CH2R4H2C + AT1002, compared to other groups. In addition, we confirmed the nontoxicity of STR-CH2R4H2C as a siRNA carrier using PAM212 cells. Thus, our results demonstrate the applicability of the combination of STR-CH2R4H2C and AT1002 for effective intradermal siRNA delivery.

FOCUS--Development of a Global Communication and Modeling Platform for Applied and Computational Medicinal Chemists.

Communication of data and ideas within a medicinal chemistry project on a global as well as local level is a crucial aspect in the drug design cycle. Over a time frame of eight years, we built and optimized FOCUS, a platform to produce, visualize, and share information on various aspects of a drug discovery project such as cheminformatics, data analysis, structural information, and design. FOCUS is tightly integrated with internal services that involve-among others-data retrieval systems and in-silico models and provides easy access to automated modeling procedures such as pharmacophore searches, R-group analysis, and similarity searches. In addition, an interactive 3D editor was developed to assist users in the generation and docking of close analogues of a known lead. In this paper, we will specifically concentrate on issues we faced during development, deployment, and maintenance of the software and how we continually adapted the software in order to improve usability. We will provide usage examples to highlight the functionality as well as limitations of FOCUS at the various stages of the development process. We aim to make the discussion as independent of the software platform as possible, so that our experiences can be of more general value to the drug discovery community.

Versatile nuclear localization signal-based oligopeptide as a gene vector.

To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (CSV40C, STR-CSV40C, and STR-CH2SV40H2C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-CSV40C and STR-CH2SV40H2C had significantly higher cellular uptake ability and transfection efficiency than SV40 and CSV40C. In particular, STR-CH2SV40H2C had higher nuclear uptake and gene expression efficiency than STR-CSV40C. Furthermore, STR-CH2SV40H2C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH2SV40H2C is a promising gene delivery system in non- or slow-dividing cells.

Prolongation of life in rats with malignant glioma by intranasal siRNA/drug codelivery to the brain with cell-penetrating peptide-modified micelles.

New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.

3D-Printed Fast-Dissolving Oral Dosage Forms via Fused Deposition Modeling Based on Sugar Alcohol and Poly(Vinyl Alcohol)-Preparation, Drug Release Studies and In Vivo Oral Absorption.

Three-dimensional printing technology holds marked promise for the pharmaceutical industry and is now under intense investigation. Most research is aimed at a greater efficiency in printing oral dosage forms using powder bed printing or fused deposition modeling (FDM). Oral dosage forms printed by FDM tend to be hard objects, which reduce the risk of cracking and chipping. However, one challenge in printing oral dosage forms via FDM is achieving rapid drug release, because the materials for FDM are basically thermoplastic polymers with slow drug release properties. In this study, we investigated printing a fast-dissolving oral dosage form by adding sugar alcohol to a poly(vinyl alcohol)-based formulation for FDM. Filaments which contain sugar alcohol were successfully prepared, and objects were printed with them as oral dosage forms by FDM. On drug release testing, a printed oral dosage form in a ring shape which contained 55% maltitol showed a more than 85% drug release in 15 min. In vivo oral absorption of this printed oral dosage form in dogs was comparable to that of a conventional fast-dissolving tablet. Of particular interest, the drug release profile and drug amount of the oral dosage forms can be easily controlled by a change in shape using 3D Computer Aided Design. These characteristics will encourage the prevalence of FDM by the pharmaceutical industry, and contribute to the promotion of personalized medicine.

Utility of a Novel Micro-Spraying Device for Intranasal Administration of Drug Solutions to Mice.

Intranasal administration has attracted attention as a means of delivering drugs because it bypasses the blood-brain barrier. However, conventional intranasal administration of drug solutions to mice using the micropipette method (MP method) is complicated and time-consuming because it requires small doses to be administered under inhalation anesthesia. This study evaluated the effectiveness of a novel intranasal administration method using Micro FPS™, a novel micro-spraying device (the MSD method). The MSD method allowed more reliable administration of the solution to the nasal mucosa than the MP method did. The transfer of inulin, a model water-soluble macromolecule compound, to the olfactory bulb and brain (cerebrum, cerebellum, brainstem, and striatum) was similar with the two methods. It also allowed the drug to be administered in a shorter time. These results suggest that the MSD method is simpler and more rapid than the MP method for intranasal administration of drugs to mice and achieves comparable delivery of inulin to the olfactory bulb and brain. Therefore, the Micro FPS™ device is a potentially useful tool for intranasal drug administration to rodents and could facilitate the development of intranasal formulations, contributing to drug development for central nervous system diseases.

Systemic delivery of siRNA to the colon using peptide modified PEG-PCL polymer micelles for the treatment of ulcerative colitis.

Ulcerative colitis (UC) is a refractory inflammatory bowel disease that causes inflammation and ulcers in the digestive tract, and significantly reduces the patient's quality of life. While existing UC treatments have many challenges, nanotechnology, and small interfering RNA (siRNA) based formulations are novel and promising for UC treatment. We previously reported that intravenous administration of MPEG-PCL-CH2R4H2C nanomicelles had high inflammatory site accumulation and remarkable therapeutic effects on rheumatoid arthritis by a phenomenon similar to enhanced permeability and retention effect. In this study, we investigated the effects of siRNA delivered using MPEG-PCL-CH2R4H2C nanomicelles through intravenous administration to the inflammation site of dextran sulfate sodium-induced colitis mice. The MPEG-PCL-CH2R4H2C micelles had optimum physical properties and high siRNA compaction ability. Moreover, model-siRNA delivered through MPEG-PCL-CH2R4H2C showed higher accumulation in the inflammatory site than that of the naked siRNA. Furthermore, intravenous administration of MPEG-PCL-CH2R4H2C/siRelA micelles, targeting siRelA, a subunit of NF-κB, significantly decreased the shortening of large intestine, clinical score, and production of inflammatory cytokines compared the 5-ASA and naked siRelA. These results suggest that MPEG-PCL-CH2R4H2C is a useful carrier for the systemic delivery and accumulation of siRNA, thus improving its therapeutic effect.

Testosterone Sustained Release Microspheres for the Treatment of Fecal Incontinence.

The objective of this study was to develop a testosterone sustained release formulation for the treatment of fecal incontinence. To suppress the initial burst release of testosterone, which can lead to systemic toxicity, we incorporated a washing step using an aminoalkyl methacrylate copolymer E solution or propylene glycol solution into a typical o/w emulsion method to prepare polylactic-co-glycolic acid microspheres. We used this method to develop a polylactic-co-glycolic acid microsphere formulation that shows sustained release of testosterone for up to one month. Not only did this formulation show a sustained release profile after administration into the intersphincteric groove in minipigs, it also increased both the anal pressure and mass of the external anal sphincter, while keeping systemic testosterone exposure low. Thus, this formulation successfully affected both the internal and external anal sphincters with a sufficient safety profile, deeming it a promising candidate treatment strategy for fecal incontinence.

Intranasal Administration of N-acetyl-L-cysteine Combined with Cell-Penetrating Peptide-Modified Polymer Nanomicelles as a Potential Therapeutic Approach for Amyotrophic Lateral Sclerosis.

Intranasal administration is a promising route for direct drug delivery to the brain; its combination with nanocarriers enhances delivery. We have previously shown that intranasal administration combined with PEG-PCL-Tat (a nanocarrier) efficiently delivers drugs to the brain and exhibits excellent therapeutic efficacy against brain diseases. We aimed to clarify whether intranasal administration combined with PEG-PCL-Tat represents a useful drug delivery system (DDS) for amyotrophic lateral sclerosis (ALS) pharmacotherapy. We used N-acetyl-L-cysteine (NAC) as a model drug with low transferability to the spinal cord and determined the physicochemical properties of NAC/PEG-PCL-Tat. After intranasal administration of NAC/PEG-PCL-Tat, we measured the survival duration of superoxide dismutase-1 G93A mutant transgenic mice (G93A mice), widely used in ALS studies, and quantitatively analyzed the tissue distribution of NAC/PEG-PCL-Tat in ddY mice. The mean particle size and zeta potential of NAC/PEG-PCL-Tat were 294 nm and + 9.29 mV, respectively. Treatment with repeated intranasal administration of NAC/PEG-PCL-Tat considerably prolonged the median survival of G93A mice by 11.5 days compared with that of untreated G93A mice. Moreover, the highest distribution after a single administration of NAC/PEG-PCL-Tat was measured in the spinal cord. These results suggest that intranasal administration combined with PEG-PCL-Tat might represent a useful DDS for ALS therapeutics.

Nose-to-brain/spinal cord delivery kinetics of liposomes with different surface properties.

The administration of liposomes via nose-to-brain delivery is expected to become a strategy for efficient drug delivery to the central nervous system. Efficient nose-to-brain delivery and the kinetics of drugs administered in this manner depend on the properties of liposomes. However, there is a lack of basic knowledge of which liposomes are suitable for this purpose. Here, a qualitative study of intranasally administered liposomes (positively charged, neutral, and negatively charged, with or without polyethylene glycol [PEG] modification; particle size <100 nm) was performed to elucidate their dynamics in the brain and spinal cord. Additionally, a quantitative investigation was performed to ascertain their distribution in each part of the brain and spinal cord. The effects of liposome surface charge and PEG modification on the kinetics and distribution post intranasal administration were investigated via two experiments. Qualitative evaluation was performed via ex vivo observation after intranasal administration of fluorescently labeled liposomes. Neutral PEG-modified liposomes were distributed throughout the brain and spinal cord 60 min after administration, and the fluorescence intensity increased with time. By contrast, non-PEG-modified neutral liposomes showed particularly strong fluorescence in the olfactory bulb, and the fluorescence was localized in the anterior part of the brain. Positively charged liposomes showed low fluorescence around the lateral part of the brain and lumbar spinal cord 60 min after administration. Low fluorescence was observed in the whole brain and spinal cord, with strong fluorescence being observed in the olfactory bulb after 120 min of administration. Negatively charged liposomes showed no fluorescence at 60 min after administration, but low fluorescence was observed throughout the brain and spinal cord 120 min after administration. We quantified the radioactivity in the brain and spinal cord after intranasal administration of radioisotope-labeled liposomes. Neutral liposomes showed the highest distribution by area under the drug concentration-time curve (AUC60-120) in the brain and spinal cord compared to other liposomes. Compared with negatively charged liposomes, positively charged liposomes had a higher distribution in the olfactory bulb and forebrain, while negatively charged liposomes had a higher distribution in the hindbrain and bulbospinal tract cord. In addition, the distribution of PEG-modified neutral liposomes in the brain and spinal cord was significantly enhanced compared to that of non-PEG-modified neutral liposomes after 90 min of intranasal administration. These results indicate that surface charge and PEG modification strongly affect the efficiency of nose-to-brain delivery kinetics, and that PEG-modified neutral liposomes are excellent carriers for drug delivery to a wide area of the brain and spinal cord.

Increased Tropism of Extracellular Vesicles Derived from Palmitic Acid-Treated Hepatocytes to Activated Hepatic Stellate Cells.

Myofibroblast-like activated hepatic stellate cells (aHSCs), which produce collagen, a major cause of liver fibrosis, are specific target cells for antifibrotic treatment. Recently, several reports have indicated that extracellular vesicles (EVs) play important roles in cell-to-cell communication through their tropism for specific cells or organs. Therefore, the present study aimed to identify aHSC-directed EVs by focusing on cell-to-cell interactions in the liver under pathological conditions. EVs were derived from the hepatocyte cell line AML12 treated with or without palmitic acid (PA) and evaluated for their physical properties and uptake by the aHSC cell line LX-2. AML12-derived EVs had a mean particle diameter of 110-130 nm, negative charge, and expressed the exosomal makers CD9 and CD63. PA-treated AML12 cells released larger EVs with higher protein levels than those without PA treatment. The intracellular uptake efficacy of EVs derived from PA-treated AML12 cells into activated LX-2 cells was significantly higher than those without PA treatment. Our study revealed that PA treatment induces hepatocytes to release EVs with aHSC-tropism. These findings may contribute to the development of an EV-based drug delivery system (DDS) for aHSC-targeted therapy and provide new insights into the role of steatotic hepatocyte-derived EVs in physiological or pathophysiological functions.

Therapeutic effects on atopic dermatitis by anti-RelA short interfering RNA combined with functional peptides Tat and AT1002.

Atopic dermatitis (AD) has high morbidity and poor prognosis because safe and effective treatments are scarce. Recently, short interfering RNA (siRNA) has shown promise as an effective treatment for targeting specific aberrantly expressed genes. However, naked siRNAs are too inefficient because of various enzymatic, membrane, and cellular barriers. We previously reported that a Tat analog acting as a cell-penetrating peptide, combined with AT1002, which reversibly increases paracellular transport of molecules across the epidermal barrier in epidermis-disrupted mice and enhances the skin permeation of water-soluble siRNA. In the present study, to develop a novel treatment for AD, we determined the intradermal permeation of siRNAs and the antiallergic effects of a siRNA that silences RelA, a member of the nuclear factor-κB family, using Tat and AT1002 peptides in an AD mouse model. We first showed that the Tat analog and AT1002 delivered siRNA into the skin of ICR mice and, upon topical application to the AD-induced ears of NC/Nga mice, changed zonula occludens protein 1 expression. In addition, the silencing effects on the mRNA of RelA in JAWS II cells transfected with siRNA oligonucleotides for mouse RelA, complexed with Tat, were as effective as a commercial vector. Furthermore, the ear thickness, clinical skin severity, topical cytokine levels, and serum IgE production in AD model mice treated with anti-RelA siRNA with Tat and AT1002 were improved.