Systems for delivering nucleic acids are now fundamental technologies for realizing personalized medicine. Among the various nucleic acid delivery systems that are currently available, ...lipid-nanoparticles (LNPs) that contain short interfering RNA (siRNA) have been extensively investigated for clinical applications. LNPs are generally prepared by an alcohol dilution method. In this method, it is necessary to remove the alcohol and then concentrate the LNP sample before they can be used. In this study, we report on the development of an “alcohol dilution–lyophilization method” for preparing siRNA-encapsulating LNPs. This method involves the use of a freeze-drying (lyophilization) method to remove the residual alcohol and to simultaneously concentrate the preparation. At first, the compositions of cryoprotectants and polyethylene glycol (PEG)-lipids that were used were optimized from the point of view of particle stabilization. A combination of sucrose and 1-(monomethoxy polyethyleneglycol5000)-2,3-dimyristoylglycerol (DMG-PEG5000) was found to have the most efficient cryoprotective activity for the LNPs. The knockdown efficiency of the LNP prepared by the alcohol dilution–lyophilization method was comparable to that of an LNP prepared by the conventional ultrafiltration method.
Iron complexes formulated as Fe(terpy)X2 (terpy = 2,2′:6′,2″-terpyridine derivatives; X = Cl, Br) were prepared and their catalytic activities for hydrosilylation of olefin with hydrosilane were ...examined. Although Fe(terpy)X2 did not show catalytic activity, the treatment of Fe(terpy)X2 with NaHBEt3 caused catalytic activity. The influence of substituents in terpy on the catalytic activity was examined, and it was found that some complexes with an unsymmetrically disubstituted terpy selectively produced 1-silylalkane with quite high catalytic activity. In the reaction of 1-octene with PhSiH3, the double-hydrosilylation product Ph(1-octyl)2SiH was selectively obtained.
This research deals with the cooperative control of CAVs (Connected and Automated Vehicles) at signal-free intersection. CAVs communicate with each other and adjust their speeds to safely pass ...through the intersection without having to stop. This is expected to improve intersection throughput and reduce fuel consumption. In order to execute cooperative control, a two-stage control structure is considered. In the first stage, the merging time, defined for each CAV as the time at which the CAV reaches the intersection, is obtained by solving mixed integer linear programming (MILP). In the second stage, each CAV solves an optimal control problem to determine the control input that allows it to reach the intersection at the merging time obtained in the first stage. In this research, a nonlinear model that considers air resistance, rolling resistance, and slope resistance is used to take into account the real environment. We propose a method using a control barrier function to guarantee the safety of CAVs even with nonlinear models , and an input correction method using a disturbance observer to suppress modelling errors caused by complex dynamics. We also propose a method that consists of establishing a speed regulation zone to avoid the possibility of passing through an intersection at an unsafe speed to improve throughput. Finally, a comparison with previous studies shows the superiority of the proposed method in reducing fuel consumption.
While the use of in vitro-transcribed mRNA (IVT-mRNA) in therapeutics is a rapidly expanding area, the transfection of the exogenous IVT-mRNA is accompanied by a risk of immune activation. This ...immunological defense mechanism suppresses cellular translation process and can reduce transfection efficiency to a considerable extent. In the present study, we investigated the in vitro effects of Integrated Stress Response Inhibitor (ISRIB), and dexamethasone, a steroidal anti-inflammatory drug, on the transfection activity of a lipid nanoparticle (LNP) that was composed of ionizable lipids and IVT-mRNA. In the case of transfection to mouse embryonic fibroblast (MEF) cells, ISRIB mainly enhanced the transfection activity at an early stage of transfection (0–6 h). In contrast, dexamethasone caused an increase in transfection activity at intermediate-late stages of transfection (4–48 h). We also investigated the in vivo effects of dexamethasone using an LNP on that the IVT-mRNA and lipid-conjugated dexamethasone (Dex-Pal) were co-loaded. The intravenous administration of the LNP successfully enhanced the protein expression in a mouse liver by up to 6.6-fold. Collectively, the co-delivery of an anti-inflammatory drug is a promising approach for enhancing transfection efficiency of IVT-mRNA.
RNA‐based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in ...the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self‐degradable lipid‐like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self‐degradability is based on a unique reaction “Hydrolysis accelerated by intra‐Particle Enrichment of Reactant (HyPER).” In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid‐scaffold lipid‐like material that mounts all of these units (ssPalmO‐Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.
Cytoplasmic release of messenger RNA from its drug delivery system is one of the key steps that determines the efficiency of protein production. Intracellular collapse of lipid nanoparticles is facilitated by the self‐degradation of ionizable lipids via a unique intraparticle reaction denoted as “Hydrolysis accelerated by intra‐Particle Enrichment of Reactant (HyPER).”
Gene therapy is a promising strategy for curing certain types of brain diseases. Supplementation of therapeutic proteins such as aromatic amino acid decarboxylase (AADC) or nerve growth factor (NGF) ...have been reported to be successful examples of such treatments. However, there are safety concerns because these systems are based on virus-based gene vectors. A safe and efficient artificial carrier is thus urgently needed as an alternative. In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As a carrier, a lipid nanoparticle (LNP) composed of environmentally sensitive lipid-like materials called an SS-cleavable proton-activated lipid-like material is used. The apolipoprotein E mediated cellular uptake of the lipid nanoparticles is one of the key features for its superior and homogeneous transfection activity compared to commercially available transfection reagents in both in vitro and in vivo situations. Immunostaining of brain specimens suggested that exogenous proteins can be introduced into neuronal cells as well as astrocytes using the mRNA-based gene carrier. This cannot be achieved using DNA-based artificial gene carriers. The findings suggest that a combination of an mRNA and a lipid based delivery system have great promise as a platform for the treatment of brain disorders.
Lymphatic endothelial cells (LECs) that form lymphatic vessels play a pivotal role in immune regulation. It was recently reported that LECs suppress the antigen-dependent anti-tumor immunity in ...cancer tissues. Thus, regulating the function of LECs is a promising strategy for cancer therapy. The objective of this study was to develop a method for the selective delivery of small interfering RNA (siRNA) to LECs. For this purpose, the siRNA was formulated into nanoparticles (LNPs) to prevent them from being degraded in body fluids and to facilitate their penetration of the cell membrane. A breakthrough technology for achieving this is ONPATTRO®, a world's first siRNA drug. Since LNPs are taken up by hepatocytes relatively well via low-density lipoprotein receptors, most of the LNP systems that have been developed so far target hepatocytes. In this study, we report on the development of a new method for the rapid and convenient method for modifying LNPs with antibodies using the CLick reaction on the Interface of the nanoParticle (CLIP). The CLIP approach was faster and more versatile than the conventional method using amide coupling. As a demonstration, we report on the LEC-targeted siRNA delivery by using antibody-modified LNPs both in vitro and in vivo. The method used for the modification of LNPs is highly promising and has the potential for expanding the LNP-based delivery of nucleic acids in the future.
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•An antibody is rapidly conjugated to an LNP via a cupper-free azide-alkyne cycloaddition reaction.•The reaction proceeds at low temperature (4 °C), even at an acidic pH, at which RNA and a pH-sensitive cationic form of the LNP is stable.•The conjugation of an anti-podoplanin antibody to an LNP significantly enhances the efficiency of siRNA delivery to lymphatic endothelial cells in the skin and in tumors.
This paper presents a case study and numerical investigation of two unstable unsaturated slopes along the Taipei Maokong Gondola system. One slope collapsed because of torrential rainfall during ...Typhoon Jangmi in September 2008, and another nearby slope developed excessive deformation under consecutive wetting and drying cycles. Recorded rainfall, measured soil parameters, site geology, and slope geometry were used in coupled hydro-mechanical finite element analyses to investigate the failure and deformation mechanisms of these two slopes. The stress paths of these two slopes were also examined and are discussed in this paper. The numerical results demonstrated that the coupled hydro-mechanical analysis satisfactorily predicted the failure and deformation characteristics of the two unstable slopes. In the collapse case, failure occurred at a shallow depth when the surficial residual soil was saturated. The slope failure was attributed to a decrease in soil shear strength when the matric suction gradually decreased as rainfall progressed. In the excessive deformation case, the numerical results suggested that pore water pressure (PWP) varied at shallow depths under wetting and drying cycles, whereas positive PWP accumulated at the soil–rock interface and induced non-uniform lateral flows parallel to the interface. The accumulated positive PWP at the interface mobilized a considerable plastic deformation in the soil. Examination of the relationships between the slope factor of safety and the corresponding hydrological data (i.e., rainfall and soil PWP) revealed a positive correlation between the slope factor of safety and accumulated rainfall. The factor of safety predicted by the infinite slope equation significantly varied with the input of PWP. This study demonstrated that the infinite slope equation using the PWP measurements obtained from the lower half of the slope could effectively predict the slope factor of safety.
•Couple hydro-mechanical analyses of two unstable unsaturated slopes are performed.•Failure mechanism and deformation characteristics of the two slopes are investigated and discussed.•The accumulated positive porewater pressure at the soil-rock interface mobilized a considerable soil plastic deformation.•A positive correlation exists between the slope factor of safety and accumulated rainfall.•The infinite slope equation using the porewater pressure obtained from the lower half of the slope could effectively predict the slope factor of safety.
Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a ...previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.
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Lipid nanoparticles (LNPs) are one of the most successful technologies in messenger RNA (mRNA) delivery. While the liver is the most frequent target for LNP delivery of mRNA, technologies for ...delivering mRNA molecules to extrahepatic tissues are also important. Herein, it is reported on the development of an LNP that targets secondary lymphoid tissues. New types of alcohol-soluble phosphatidylserine (PS) derivatives are designed as materials that target immune cells and then incorporated into LNPs using a microfluidic technique with a high degree of scalability and reproducibility. The resulting LNP that contained the synthesized PS delivered mRNA to the spleen much more efficiently compared to a control LNP. A sub-organ analysis revealed that the PS-loaded LNP is extensively taken up by tissue-resident macrophages in the red pulp and the marginal zone of the spleen. Thus, the PS-loaded LNP reported in this study will be a promising strategy for clinical applications that involve delivering mRNA to the spleen.
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