Hepatocyte-Targeted Delivery of siRNA Polyplex with PEG-Modified Lactosylated Dendrimer/Cyclodextrin Conjugates for Transthyretin-Related Amyloidosis Therapy.

Targeted drug delivery system (DDS) is required for RNA interference (RNAi) therapy to increase the therapeutic effect and to reduce the adverse effect. Especially in transthyretin (TTR)-related amyloidosis, hepatocyte specific delivery is desired because TTR mainly expresses in hepatocyte. Herein, we report on a hepatocyte-specific small interfering RNA (siRNA) delivery system using polyethylene glycol (PEG)-modified lactosylated dendrimer (generation 3; G3) conjugates with α-cyclodextrin (PEG-LαCs (G3)) for TTR-related amyloidosis therapy, and investigated the in vitro and in vivo gene silencing effect of PEG-LαCs (G3)/siRNA polyplexes. PEG-LαC (G3, average degree of substitution of PEG (DSP) 2)/TTR siRNA (siTTR) polyplex exhibited the asialoglycoprotein receptor (ASGPR)-mediated cellular uptake, high endosomal escaping ability and localization of the siRNA in cytoplasm, resulting in significant TTR silencing in HepG2 cells. In vivo studies showed that PEG-LαC (G3, DSP2)/siTTR polyplex led to a significant TTR silencing effect in liver after systemic administration to mice. Furthermore, safety evaluation revealed that PEG-LαC (G3, DSP2)/siTTR polyplex had no significant toxicity both in vitro and in vivo. These findings suggest the utility of PEG-LαC (G3, DSP2) as a promising hepatocyte-specific siRNA delivery system both in vitro and in vivo, and as a therapeutic approach for TTR-related amyloidosis.

Recent Advances in Oligonucleotide-Based Therapy for Transthyretin Amyloidosis: Clinical Impact and Future Prospects.

Transthyretin (TTR) amyloidosis, also known as transthyretin-related familial amyloidotic polyneuropathy (ATTR-FAP), is a fatal hereditary systemic amyloidosis caused by mutant forms of TTR. Although conventional treatments for ATTR-FAP, such as liver transplantation (LT) and TTR tetramer stabilizer, reportedly halt the progression of clinical manifestation, these therapies have several limitations. Oligonucleotide-based therapy, e.g. small interfering RNA (siRNA)- and antisense oligonucleotides (ASOs)-based therapy, hold enormous potential for the treatment of intractable diseases such as ATTR-FAP, by specifically regulating the gene responsible for the disease. Clinical evidence strongly suggests that LT inhibits mutant TTR production, thus improving the manifestation of ATTR-FAP. Therefore, an oligonucleotide-based therapy for ATTR-FAP, which reduces the production of TTR by the liver, has recently been developed in preclinical and clinical studies. This review focuses on recent advances in oligonucleotide-based therapy and future prospects of next-generation oligonucleotide-based drugs for therapeutic use against ATTR-FAP.

Species differences take shape at nanoparticles: protein corona made of the native repertoire assists cellular interaction.

Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell-nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.

Time-course profiling of molecular stress responses to silver nanoparticles in the earthworm Eisenia fetida.

The molecular mechanism of silver nanoparticle (AgNP) toxicity, particularly its temporal aspect, is currently limited in the literature. This study seeks to identify and profile changes in molecular response patterns over time during soil exposure of the earthworm Eisenia fetida to AgNPs (82±27 nm) with reference to dissolved silver salt (AgNO3). Principal component analysis of selected gene and enzyme response profiles revealed dissimilar patterns between AgNO3 and AgNP treatments and also over time. Despite the observed difference in molecular profiles, the body burdens of total Ag were within the same range (10-40 mg/kg dry weight worm) for both treatments with apparent correlation to the induction pattern of metallothionein. AgNO3 induced the genes and enzymes related to oxidative stress at day 1, after which markers of energy metabolism were all suppressed at day 2. Exposure to AgNPs likewise led to induction of oxidative stress genes at day 2, but with a temporal pattern shift to immune genes at day 14 following metabolic upregulation at day 7. The involvement of oxidative stress and subsequent alterations in immune gene regulation were as predicted by our in vitro study reported previously, highlighting the importance of immunological endpoints in nanosilver toxicity.

Potential use of lactosylated dendrimer (G3)/α-cyclodextrin conjugates as hepatocyte-specific siRNA carriers for the treatment of familial amyloidotic polyneuropathy.

To reveal the potential use of lactosylated-dendrimer (G3) conjugates with α-cyclodextrin (Lac-α-CDE (G3)) as novel hepatocyte-specific siRNA carriers in order to treat transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP), we evaluated the RNAi effect of siRNA complexes with Lac-α-CDE (G3) both in vitro and in vivo. Herein, we targeted TTR gene expression because TTR-related FAP was often caused by amyloidogenic TTR (ATTR), which mainly expresses in hepatocytes. Lac-α-CDE (G3, average degree of substitution of lactose (DSL) 1.2)/siRNA complex had a potent RNAi effect against TTR gene expression through adequate physicochemical properties, asialoglycoprotein receptor (ASGP-R)-mediated cellular uptake, efficient endosomal escape and the delivery of the siRNA complex to cytoplasm, but not nucleus, with negligible cytotoxicity. Lac-α-CDE (G3, DSL 1.2)/siRNA complex had the potential to induce the in vivo RNAi effect after intravenous administration in the liver of mice. The blood chemistry values in the α-CDE (G3) and Lac-α-CDE (G3, DSL 1.2) systems were almost equivalent to those in the control system (5% mannitol solution). Taken together, these results suggest that Lac-α-CDE (G3, DSL 1.2) has the potential for a novel hepatocyte-selective siRNA carrier in vitro and in vivo, and has a possibility as a therapeutic tool for FAP to the liver transplantation.

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles.

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid ß, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid ß deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst.

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.

Surface modification effects of graphite for selective hydrogen absorption by titanium at room temperature.

Surface modification effects of graphite and organic solvents on Ti were investigated by thermogravimetry (TG), Raman spectroscopy, and transmission electron microscopy (TEM) observations to improve its hydrogen absorption properties. As a result, Ti ball-milled with graphite showed high reactivity and selectivity for hydrogen with high durability.

Nanomaterials and Annelid Immunity: A Comparative Survey to Reveal the Common Stress and Defense Responses of Two Sentinel Species to Nanomaterials in the Environment.

Earthworms and leeches are sentinel animals that represent the annelid phylum within terrestrial and freshwater ecosystems, respectively. One early stress signal in these organisms is related to innate immunity, but how nanomaterials affect it is poorly characterized. In this survey, we compare the latest literature on earthworm and leeches with examples of their molecular/cellular responses to inorganic (silver nanoparticles) and organic (carbon nanotubes) nanomaterials. A special focus is placed on the role of annelid immunocytes in the evolutionarily conserved antioxidant and immune mechanisms and protein corona formation and probable endocytosis pathways involved in nanomaterial uptake. Our summary helps to realize why these environmental sentinels are beneficial to study the potential detrimental effects of nanomaterials.

Tracing the In Vivo Fate of Nanoparticles with a "Non-Self" Biological Identity.

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association.

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.

Differential Nanoparticle Sequestration by Macrophages and Scavenger Endothelial Cells Visualized in Vivo in Real-Time and at Ultrastructural Resolution.

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.

Self-assembled monolayer electrode of a diiron complex with a phenoxo-based dinucleating ligand: observation of molecular oxygen adsorptionldesorption in aqueous media.

The phenoxo-based dinucleating ligand, 2,6-bis[bis(6-pivalamido-2-pyridylmethyl)amino-methyl-4-aminophenol (1), and its Fe2(II) complex, [Fe2(II)(1)(PhCOO)2](CF3SO3) (2), were prepared and 2 deposited on the Au surface (2/Au) is much more stable than in solution and exhibits redox behavior in aqueous media as well as reversible adsorption/desorption of oxygen at room temperature.

In vitro and in vivo siRNA delivery to hepatocyte utilizing ternary complexation of lactosylated dendrimer/cyclodextrin conjugates, siRNA and low-molecular-weight sacran.

In this study, we newly developed the ternary complexes consisting of lactosylated dendrimer (generation 3)/α-cyclodextrin conjugate (Lac-α-CDE), siRNA and the anionic polysaccharide sacrans, and evaluated their utility as siRNA transfer carriers. Three kinds of the low-molecular-weight sacrans, i.e. sacran (100) (Mw 44,889Da), sacran (1000) (Mw 943,692Da) and sacran (10,000) (Mw 1,488,281Da) were used. Lac-α-CDE/siRNA/sacran ternary complexes were prepared by adding the low-molecular-weight sacrans to the Lac-α-CDE/siRNA binary complex solution. Cellular uptake of the ternary complex with sacran (100) was higher than that of the binary complex or the other ternary complexes with sacran (1000) and sacran (10,000) in HepG2 cells. Additionally, the ternary complex possessed high serum resistance and endosomal escaping ability in HepG2 cells. High liver levels of siRNA and Lac-α-CDE were observed after the intravenous administration of the ternary complex rather than that of the binary complex. Moreover, intravenous administration of the ternary complex (siRNA 5mg/kg) induced the significant RNAi effect in the liver of mice with negligible change of blood chemistry values. Therefore, a ternary complexation of the Lac-α-CDE/siRNA binary complex with sacran is useful as a hepatocyte-specific siRNA delivery system.

A clue to unprecedented strategy to HIV eradication: "Lock-in and apoptosis".

Despite the development of antiretroviral therapy against HIV, eradication of the virus from the body, as a means to a cure, remains in progress. A "kick and kill" strategy proposes "kick" of the latent HIV to an active HIV to eventually be "killed". Latency-reverting agents that can perform the "kick" function are under development and have shown promise. Management of the infected cells not to produce virions after the "kick" step is important to this strategy. Here we show that a newly synthesized compound, L-HIPPO, captures the HIV-1 protein Pr55Gag and intercepts its function to translocate the virus from the cytoplasm to the plasma membrane leading to virion budding. The infecting virus thus "locked-in" subsequently induces apoptosis of the host cells. This "lock-in and apoptosis" approach performed by our novel compound in HIV-infected cells provides a means to bridge the gap between the "kick" and "kill" steps of this eradication strategy. By building upon previous progress in latency reverting agents, our compound appears to provide a promising step toward the goal of HIV eradication from the body.

Neuronal sFlt1 and Vegfaa determine venous sprouting and spinal cord vascularization.

Formation of organ-specific vasculatures requires cross-talk between developing tissue and specialized endothelial cells. Here we show how developing zebrafish spinal cord neurons coordinate vessel growth through balancing of neuron-derived Vegfaa, with neuronal sFlt1 restricting Vegfaa-Kdrl mediated angiogenesis at the neurovascular interface. Neuron-specific loss of flt1 or increased neuronal vegfaa expression promotes angiogenesis and peri-neural tube vascular network formation. Combining loss of neuronal flt1 with gain of vegfaa promotes sprout invasion into the neural tube. On loss of neuronal flt1, ectopic sprouts emanate from veins involving special angiogenic cell behaviours including nuclear positioning and a molecular signature distinct from primary arterial or secondary venous sprouting. Manipulation of arteriovenous identity or Notch signalling established that ectopic sprouting in flt1 mutants requires venous endothelium. Conceptually, our data suggest that spinal cord vascularization proceeds from veins involving two-tiered regulation of neuronal sFlt1 and Vegfaa via a novel sprouting mode.

Single oxygen generation sensitized by spiro(dipyridinogermole)(dithienogermole)s.

Three spiro(dipyridinogermole)(dithienogermole) derivatives (1-3), including newly prepared spiro(dipyridinogermole)[di(2-pyridyl)dithienogermole] (3), were examined as photosensitizers for singlet oxygen (1O2) generation in dichloromethane-methanol. Irradiation of their air-saturated solutions led to the generation of 1O2, which was readily trapped by well-known scavengers, dihydronaphthoquinone (DHN) and diphenylisobenzofuran (DPBF). Spiro(dipyridinogermole)[bis(n-hexylbithiophenyl)dithienogermole] (2) showed the best performance with a first-order rate constant that was higher than that of tetraphenylporphyrin (TPP), an efficient photosensitizer for 1O2 generation. This is ascribable to the efficient intersystem crossing characteristic of the dipyridinogermole unit. The quantum yield of 1O2 generation was φΔ = 0.72 for 2, relative to that for rose bengal (RB) in methanol as reference (φΔ = 0.8).

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona.

Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells' protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.