L-Type Calcium Channel Blocker Enhances Cellular Delivery and Gene Silencing Potency of Cell-Penetrating Asymmetric siRNAs.

The efficient delivery of small interfering RNAs (siRNAs) to the target cells is critical for the pharmaceutical success of RNA interference (RNAi) drugs. One of the possible strategies to improve siRNA delivery is to identify auxiliary molecules that augment their cellular uptake. Herein, we performed a chemical library screening in an effort to discover small molecules that enhance the potency of cholesterol-conjugated, cell-penetrating asymmetric siRNAs (cp-asiRNAs). Interestingly, three compounds identified from the screen share a common dihydropyridine (DHP) core and function as L-type calcium channel blockers (CCBs). Using confocal microscopy and quantitative analysis of small RNAs, we demonstrated that the L-type CCBs increased the endocytic cellular uptake of cp-asiRNAs. Furthermore, these small molecules substantially improved the potency of cp-asiRNAs, not only in vitro but also in vivo on rat skin. Collectively, our study provides an alternative pharmacological approach for the identification of small molecules that potentiate the effects of therapeutic siRNAs.

The natural phytochemical trans-communic acid inhibits cellular senescence and pigmentation through FoxO3a activation.

Ageing is characterized by the accumulation of chronic and irreversible oxidative damage, chronic inflammation and organ dysfunction. To attenuate these ageing-related changes, various natural phytochemicals are often applied. Trans-communic acid (TCA), an active component of brown pine leaf extract, has antimicrobial and cancer chemopreventive activity and inhibits ultraviolet B (UVB)-induced MMP-1 expression. To determine whether the phytochemical TCA could affect the lifespan of an ageing model, Caenorhabditis elegans prevent ageing-related phenotypes of the skin. Caenorhabditis elegans (C. elegans) wild-type N2 and mutant strains were used in this study to explore the lifespan extension effect of TCA and its mechanism. We estimated lipofuscin accumulation and melanin levels, which are closely associated with skin senescence. Moreover, we explored the mechanism of action associated with ageing attenuation. We performed oxidative stress resistance and thermotolerance assays in C. elegans and surface plasmon resonance analysis of TCA binding with the forkhead box-O3a (FoxO3a) protein. TCA, which is the active component in Korean red pine (Pinus densiflora), attenuated ageing-related changes in skin cells. TCA lowered lipofuscin accumulation in fibroblasts and decreased melanin levels in melanocytes. These protective effects were mediated by activation of the representative longevity gene FoxO3a, which was induced by direct binding with TCA. Interestingly, TCA extended the lifespan of C. elegans, although it did not affect stress resistance, oxidative stress or thermotolerance. These results strongly suggest that TCA prevents the senescent phenotype of model organisms and exhibits beneficial effects on ageing-related skin phenotypes through direct FoxO3a activation.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents.

BACKGROUND/AIMS: Dehydroabietic acid (DAA) is a natural phytochemical found in red pine trees and herbal plants. While DAA and its derivatives are known for improving diabetes and hyperlipidemia, the antiaging effect and its underlying mechanisms of DAA on skin have not been fully examined. Here, we assessed the antiaging effects of DAA on human dermal fibroblasts and skin equivalents. METHODS: We investigated the effect of DAA on the secretion of type I procollagen and matrix metalloproteinase-1 (MMP-1) in ultraviolet B (UVB)-irradiated neonatal normal human dermal fibroblasts (NHDFn). Using nonlinear optical imaging techniques, we visualized quantitative and qualitative changes of collagen fibers by DAA treatment in human skin equivalent models. RESULTS: DAA induces increases in type I procollagen secretion when treated on UVB-irradiated NHDFn. DAA also downregulates secretion of MMP-1 through the inhibition of the JNK signaling pathway. In human skin equivalent models, we successfully visualized the spatial distribution of collagen fibers in the dermis and found that quantity, diameter, and arrangement of collagen fibers in the dermis were significantly improved by DAA treatment. CONCLUSION: Our results suggest that DAA could be a useful agent for improving skin photoaging through the protection and regeneration of collagen fibers in skin.

Dehydroabietic Acid Suppresses Inflammatory Response Via Suppression of Src-, Syk-, and TAK1-Mediated Pathways.

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid derived from coniferous plants such as Pinus and Picea. Various bioactive effects of DAA have been studied including antibacterial, antifungal, and anticancer activities. However, the anti-inflammatory mechanism of DAA remains unclear. We evaluated the anti-inflammatory effect of DAA in macrophage cell lines. Dehydroabietic acid clearly reduced nitric oxide (NO) production and inflammatory gene expression decreased according to RT-PCR results. Dehydroabietic acid displayed anti-inflammatory activity at the transcriptional level in results from NF-κB- or AP-1-mediated luciferase assays. To identify the DAA target protein, we investigated NF-κB and AP-1 pathways by Western blotting analysis. Dehydroabietic acid suppressed the activity of proto-oncogene tyrosine protein kinase (Src) and spleen tyrosine kinase (Syk) in the NF-κB cascade and transforming growth factor beta-activated kinase 1 (TAK1) in the AP-1 cascade. Using overexpression strategies, we confirmed that DAA targeted these kinases. Our findings demonstrate the anti-inflammatory effects and molecular mechanism of DAA. This suggests that DAA has potential as a drug or supplement to ameliorate inflammation.

In Vitro Effects of Dehydrotrametenolic Acid on Skin Barrier Function.

Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.

The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells.

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

Transcriptomic variation in proanthocyanidin biosynthesis pathway genes in soybean (Glycine spp.).

BACKGROUND: Proanthocyanidins are oligomeric or polymeric end products of flavonoid metabolic pathways starting with the central phenylpropanoid pathway. Although soybean (Glycine spp.) seeds represent a major source of nutrients for the human diet, as well as components for the cosmetics industry as a result of their high levels of flavonoid metabolites, including isoflavonoids, anthocyanins and proanthocyanidins, the genetic regulatory mechanisms underlying proanthocyanidin biosynthesis in soybean remain unclear. RESULTS: We evaluated interspecific and intraspecific variability in flavonoid components in soybean using 43 cultivars, landraces and wild soybean accessions. We performed transcriptomic profiling of genes encoding enzymes involved in flavonoid biosynthesis using three soybean genotypes, Hwangkeum (elite cultivar), IT109098 (landrace) and IT182932 (wild accession), in seeds. We identified a Glycine max landrace, IT109098, with a proanthocyanidin content as high as that of wild soybean. Different homologous genes for anthocyanidin reductase, which is involved in proanthocyanidin biosynthesis, were detected as differentially expressed genes between IT109098 and IT182932 compared to Hwangkeum. CONCLUSION: We detected major differences in the transcriptional levels of genes involved in the biosynthesis of proanthocyanidin and anthocyanin among genotypes beginning at the early stage of seed development. The results of the present study provide insights into the underlying genetic variation in proanthocyanidin biosynthesis among soybean genotypes. © 2017 Society of Chemical Industry.

Licochalcone A, a polyphenol present in licorice, suppresses UV-induced COX-2 expression by targeting PI3K, MEK1, and B-Raf.

Licorice is a traditional botanical medicine, and has historically been commonly prescribed in Asia to treat various diseases. Glycyrrhizin (Gc), a triterpene compound, is the most abundant phytochemical constituent of licorice. However, high intake or long-term consumption of Gc has been associated with a number of side effects, including hypertension. However, the presence of alternative bioactive compounds in licorice with anti-carcinogenic effects has long been suspected. Licochalcone A (LicoA) is a prominent member of the chalcone family and can be isolated from licorice root. To date, there have been no reported studies on the suppressive effect of LicoA against solar ultraviolet (sUV)-induced cyclooxygenase (COX)-2 expression and the potential molecular mechanisms involved. Here, we show that LicoA, a major chalcone compound of licorice, effectively inhibits sUV-induced COX-2 expression and prostaglandin E2 PGE2 generation through the inhibition of activator protein 1 AP-1 transcriptional activity, with an effect that is notably more potent than Gc. Western blotting analysis shows that LicoA suppresses sUV-induced phosphorylation of Akt/ mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinases (ERK)1/2/p90 ribosomal protein S6 kinase (RSK) in HaCaT cells. Moreover, LicoA directly suppresses the activity of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK)1, and B-Raf, but not Raf-1 in cell-free assays, indicating that PI3K, MEK1, and B-Raf are direct molecular targets of LicoA. We also found that LicoA binds to PI3K and B-Raf in an ATP-competitive manner, although LicoA does not appear to compete with ATP for binding with MEK1. Collectively, these results provide insight into the biological action of LicoA, which may have potential for development as a skin cancer chemopreventive agent.

Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

RIG-I-Mediated Innate Immune Stimulation by Chemically Synthesized Long Double-Stranded RNAs Is Structure and Sequence Dependent.

Double-stranded RNAs (dsRNAs) longer than 30 bp have not been considered desirable RNA interference (RNAi) triggering structures in mammalian cells as they nonspecifically activate innate immune response. However, in earlier studies, not only dsRNA length but also 5'-triphosphate moiety produced by in vitro transcription might have affected the stimulation of innate immune system. Herein, using chemically synthesized long dsRNAs without 5'-triphosphate, we elucidated direct relationship between length of dsRNAs and innate immune stimulation. First, we found that blunt-ended, chemically synthesized 38/40-60 bp-long dsRNAs induced retinoic acid-inducible gene I (RIG-I)-mediated innate immune response, which was suppressed by the introduction of the 2-nt 3' overhang structure. Surprisingly, we discovered that RIG-I activation by these long dsRNAs is also sequence dependent, and the sequence composition at dsRNA termini is important for RIG-I activation. In addition, we identified that long dsRNAs over 38 bp could elicit single- or dual-target gene silencing in a Dicer-independent manner. Taken together, our findings may serve as guidelines to develop an immunostimulatory RNAi trigger to exploit host's innate immune system, as well as a specific dual-gene targeting RNAi therapeutics platform without nonspecific innate immune stimulation by RIG-I.

5,7-dimethoxyflavone induces melanogenesis in B16F10 melanoma cells through cAMP-dependent signalling.

Melanin protects the skin against ultraviolet radiation (UVR) and diverse free radicals. Agents that increase melanin synthesis in melanocytes may reduce UVR-induced skin damage and skin cancer. In the present study, we evaluated the effects of 5,7-dimethoxyflavone (5,7-DMF) on melanogenic protein expression and signalling pathways. We found that 5,7-DMF significantly increased melanin content by upregulating microphthalmia-associated transcription factor and related melanogenic proteins. Additionally, 5,7-DMF increased cAMP levels, which activates a cascade of reactions, such as cAMP responsive element-binding protein and Akt/glycogen synthase kinase-3ß (GSK-3ß) signalling. Thus, 5,7-DMF may be an effective pigmentation stimulator for photoprotection and hypopigmentation disorders.

Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E2 (PGE2) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE2 secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE2 secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

A novel mineralocorticoid receptor antagonist, 7,3',4'-trihydroxyisoflavone improves skin barrier function impaired by endogenous or exogenous glucocorticoids.

Excess glucocorticoids (GCs) with either endogenous or exogenous origins deteriorate skin barrier function. GCs bind to mineralocorticoid and GC receptors (MRs and GRs) in normal human epidermal keratinocytes (NHEKs). Inappropriate MR activation by GCs mediates various GC-induced cutaneous adverse events. We examined whether MR antagonists can ameliorate GC-mediated skin barrier dysfunction in NHEKs, reconstructed human epidermis (RHE), and subjects under psychological stress (PS). In a preliminary clinical investigation, topical MR antagonists improved skin barrier function in topical GC-treated subjects. In NHEKs, cortisol induced nuclear translocation of GR and MR, and GR and MR antagonists inhibited cortisol-induced reductions of keratinocyte differentiation. We identified 7,3',4'-trihydroxyisoflavone (7,3',4'-THIF) as a novel compound that inhibits MR transcriptional activity by screening 30 cosmetic compounds. 7,3',4'-THIF ameliorated the cortisol effect which decreases keratinocyte differentiation in NHEKs and RHE. In a clinical study on PS subjects, 7,3',4'-THIF (0.1%)-containing cream improved skin barrier function, including skin surface pH, barrier recovery rate, and stratum corneum lipids. In conclusion, skin barrier dysfunction owing to excess GC is mediated by MR and GR; thus, it could be prevented by treatment with MR antagonists. Therefore, topical MR antagonists are a promising therapeutic option for skin barrier dysfunction after topical GC treatment or PS.

Metabolomic understanding of pod removal effect in soybean plants and potential association with their health benefit.

Since natural materials, such as phytochemicals in plants, are increasingly being used for foods and skincare due to their beneficial functions, it is important for developing the cultivation practices to increase the contents of phytochemicals. We here explored metabolite perturbations in the leaves of soybean plants when their pods were removed during growth through 1H NMR-based metabolomics approach. There were obvious metabolic differences in the leaves between normal and pod-removed soybean plants. High amounts of primary metabolites in pod-removed soybean leaves, including amino acids, sugars, and fatty acids, reflected a delay of leaf senescence caused by pod removal. In particular, amounts of isoflavones, coumestrol, and apigenin derivatives in pod-removed soybean leaves were substantially increased. These were considered as distinct metabolic influences of pod removal in soybean plants. These results indicate that pod removal of soybean plants can induce significant perturbations of various metabolites in their soybean leaves, providing useful information to improve the quality of soybean leaves by increasing amounts of bioactive components.

Metabotyping of different soybean genotypes and distinct metabolism in their seeds and leaves.

The metabolome of three soybean genotypes, Glycine max Hwangkeum (elite or domesticated cultivar), Glycine max Napjakong (landrace or semi-wild cultivar) and Glycine soja Dolkong (wild cultivar), were characterized in seeds and leaves using a 1H NMR-based metabolomics approach. Expression of primary and secondary metabolites were different in seeds and leaves as well as amongst soybean genotypes. Different kaempferol glycosides were observed in the leaves but not in the seeds, and quercetin derivatives were found only in G. max Napjakong and G. soja Dolkong. Moreover, epicatechin was found only in the seeds of G. max Napjakong and G. soja Dolkong. These results demonstrate distinct adaptations of different soybean genotypes to given environmental conditions. The current study, therefore, provides useful information on global metabolic compositions that might be used to develop soybean-based products through better understanding of the metabolic phenotypes of existing soybean genotypes.

Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages.

Cytokines and chemokines are transcriptionally regulated by inflammatory transcription factors such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor (IRF)-3. A daidzein derivative compound, 8-hydroxydaidzein (8-HD), isolated from soy products, has recently gained attention due to various pharmacological benefits, including anti-inflammatory activities. However, regulation of the inflammatory signaling mechanism for 8-HD is still poorly understood, particularly with respect to the IRF-3 signaling pathway. In this study, we explored the molecular mechanism of 8-HD in regulating inflammatory processes, with a focus on the IRF-3 signaling pathway using a lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [Poly (I:C)] stimulated murine macrophage cell line (RAW264.7). The 8-HD downregulated the mRNA expression level of IRF-3-dependent genes by inhibiting phosphorylation of the IRF-3 transcription factor. The inhibitory mechanism of 8-HD in the IRF-3 signaling pathway was shown to inhibit the kinase activity of IKKε to phosphorylate IRF-3. This compound can also interfere with the TRIF-mediated complex formation composed of TRAF3, TANK, and IKKε leading to downregulation of AKT phosphorylation and reduction of IRF-3 activation, resulted in inhibition of IRF-3-dependent expression of genes including IFN-ß, C-X-C motif chemokine 10 (CXCL10), and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Therefore, these results strongly suggest that 8-HD can act as a promising compound with the regulatory function of IRF-3-mediated inflammatory responses.

Tripodal RNA with 3' Overhangs Prevents RIG-I-Mediated Innate Immune Response.

RNA interference (RNAi) offers great promise in life science research and therapeutic development, as it easily achieves a potent target gene knockdown with high specificity. Since the conventional small interfering RNA (siRNA) structure, known as 19 bp double-stranded RNA (dsRNA) with 2-nucleotide (nt) 3' overhang, has been introduced to successfully elicit the RNAi in mammalian cells, a variety of structural variants of RNAi trigger have been developed. Our group previously reported branched, tripodal interfering RNA (tiRNA) structures as a multigene targeting RNA structure inducing RNAi. However, the immune stimulatory effect of branched tiRNA structure has not been thoroughly evaluated. In this study, we show that tiRNA with blunt ends triggers innate immune response in T98G cell and mouse macrophage cells, which is dependent upon the retinoic acid-inducible gene I (RIG-I), a well-known cytoplasmic dsRNA sensor. Interestingly, immune response triggered by tiRNA can be suppressed by the introduction of 2-nt 3' overhang structure. Our finding expands the structural diversity of RIG-I ligands and provides a guide to develop a safe multitargeting RNA structure for therapeutic application.

Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen.

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMS content is controlled by multiple genetic factors, the genetic basis of CMS biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMS biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMS. Two of the other genes, annotated as encoding a MYB domain protein and a MLP-like protein, may increase CMS accumulation in response to stress conditions. Our results will help to complete our understanding of the CMS biosynthetic pathway, and should facilitate development of soybean cultivars with high CMS content that could be used to promote the fitness of plants and human beings.

Multi-block magnetic nanorods for controlled drug release modulated by Fourier transform surface plasmon resonance.

Stimuli-responsive tunable drug release using nanocarriers is an important subject in smart drug delivery systems. Specifically, magnetic-responsive nanocarriers provide a great opportunity for remote control as well as on-demand command. To effectively utilize magnetic-responsive nanocarriers in vivo and in vitro, drug release should not only be controlled in an efficient way, but also monitored in situ. To satisfy those prerequisites, a template-assisted electrochemical deposition method can be a great option for the synthesis of designer materials that are targeted for specific purposes. Here, we synthesized plasmonic-magnetic nanocarriers by template-assisted electrochemical deposition and covered their surface with a silica shell for drug loading. By appropriately designing the blocks, we synthesized nanocarriers that were plasmonically active and magnetically active with spaces for drug payload. These nanocarriers could be modulated under an external magnetic field and their rotation (or agitation) could be monitored by Fourier transform conversion. Using our nanocarriers, we systematically investigated the tunable release of the anticancer drug doxorubicin as a function of the external magnetic field. Additionally, by applying this modulation system to an in vitro system using HeLa cells we were able to not only monitor the modulation systems but also tailor the drug release in a controlled manner. We expect that our approach will contribute to understanding of nanocarriers in a simulative manner in vitro.

RNA Interference-Mediated Gene Silencing by Branched Tripodal RNAs Does Not Require Dicer Processing.

Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.