The efficacy of mRNA-lipid nanoparticles (mRNA-LNPs) depends on several factors, including their size and morphology. This study presents a new technique to characterize mRNA-LNPs in an aqueous ...medium using atomic force microscopy (AFM). This method utilizes an anti-polyethylene glycol antibody to immobilize mRNA-LNPs onto a glass substrate without corruption, which cannot be avoided with conventional procedures using solid substrates such as mica and glass. The obtained AFM images showed spherical and bleb-like structures of mRNA-LNPs, consistent with previous observations made using cryo-transmission electron microscopy. The AFM method also revealed the predominant existence of nanoparticles with a diameter < 60 nm, which were not detectable by dynamic light scattering and nanoparticle tracking analysis. As mRNA-LNPs are usually not monodisperse, but rather polydisperse, the AFM method can provide useful complementary information about mRNA-LNPs in their development and quality assessment.
The occurrence of complex coacervation in an aqueous mixture of proteins (lysozyme, albumin, immunoglobulin G) and hyaluronic acid and its effect on protein transition in a model system was studied ...to elucidate factors determining the bioavailability of subcutaneously injected therapeutic proteins. Mixing of hyaluronic acid and the model proteins induced complex coacervation at solution pH close to or below the isoelectric point of the proteins. In vitro dialysis using membranes with large pore size tube represented a limitation in the protein transition of the coacervation mixture. Thermal analysis suggested there was retention of the protein conformation in the polymer complex.
The purpose of this study was to elucidate the effect of high-temperature storage on the stability of ranitidine, specifically with respect to the potential formation of N-nitrosodimethylamine ...(NDMA), which is classified as a probable human carcinogen. Commercially available ranitidine reagent powders and formulations were stored under various conditions, and subjected to LC-MS/MS analysis. When ranitidine tablets from two different brands (designated as tablet A and tablet B) were stored under accelerated condition (40 °C with 75% relative humidity), following the drug stability guidelines issued by the International Conference on Harmonisation (ICH-Q1A), for up to 8 weeks, the amount of NDMA in them substantially increased from 0.19 to 116 ppm and from 2.89 to 18 ppm, respectively. The formation of NDMA that exceeded the acceptable daily intake limit (0.32 ppm) at the temperature used under accelerated storage conditions clearly highlights the risk of NDMA formation in ranitidine formulations when extrapolated to storage under ambient conditions. A forced-degradation study under the stress condition (60 °C for 1 week) strongly suggested that environmental factors such as moisture and oxygen are involved in the formation of NDMA in ranitidine formulations. Storage of ranitidine tablets and reagent powders at the high temperatures also increased the amount of nitrite, which is considered one of the factors influencing NDMA formation. These data indicate the necessity of controlling/monitoring stability-related factors, in addition to controlling impurities during the manufacturing process, in order to mitigate nitrosamine-related health risks of certain pharmaceuticals.
Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane ...penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.
The relationship between the geometric particle size distribution (GPSD) and the aerodynamic particle size distribution (APSD) of commercial solution and suspension metered-dose inhaler (MDI) ...formulations was assessed to clarify the use of GPSD to estimate the APSD. The size distribution of particles discharged from four suspension and four solution MDIs was measured using the Inas®100 light-scattering spectrometer and a Next Generation Impactor. The conversion factor was calculated by measuring the GPSD and APSD of MDIs. The morphology and physical properties of MDIs were studied using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Six of the eight MDIs showed similar conversion factor profiles, irrespective of their composition and formulation types. Applying the conversion factor obtained from one of the six MDIs resulted in a particle size distribution comparable to each APSD except for some formulations. The two other solution MDIs, which contained citric acid, had much higher and variable conversion factors. SEM images and DSC scans of the solids obtained by nebulization of the solutions containing beclomethasone and/or citric acid showed the formation of a paste-like amorphous solid. These results indicated that APSD of solution and suspension MDIs that form rigid particles may be estimated by using the conversion factor and GPSD. Contrarily, the estimation is more difficult in formulations that tend to lose the particle structure during the measurement.
Achieving appropriate inhalation in patients with coronavirus disease 2019 (COVID-19) is a common challenge in the use of repurposed metered-dose inhaler (MDI) formulations. The purpose of this study ...was to evaluate the effect of five valved holding chambers (VHCs) on the inhalation of ciclesonide from Alvesco MDI. The aerodynamic particle size distribution of ciclesonide discharged from Alvesco MDI was evaluated using a Next Generation Impactor in the presence and absence of VHCs. The use of VHCs retained or slightly increased the amount of ciclesonide in the fine particle diameter range (aerodynamic particle size below 3 μm) (FPD) and reduced the amount at the induction port after coordinated inhalation. However, the use of VHC reduced the FPD of the formulation by increasing the time between the MDI discharge and the pump suction by various degrees among the five VHCs. These results indicated that use of the VHCs and minimizing the inhalation delay time should ensure sufficient inhalation of ciclesonide particles.
From examination of the central axonal projections of sensory bristles on the notum of several species of Drosophilidae, we demonstrate different features that may indicate different functions for ...macro- and microchaetes. The large macrochaetes have conserved arborizations that correlate with their conserved position. Nevertheless, we find evidence for only two discrete projection patterns for bristles in the dorsocentral (DC) row, even when there may be four or five bristles present. We show that the small microchaetes of
Drosophila melanogaster display regional specificity and subsets of contiguous bristles project to a common region in the thoracic ganglion. Interestingly, the axons of each of these subsets also form a specific fasciculation group on the scutum before joining the axon of a particular macrochaete. The positions of microchaetes on the scutum and the shape of the fasciculation groups vary between closely related species. There is no correlation between body size, bristle patterns, and fasciculation patterns. Furthermore, none of these traits correlate with the phylogenetic relationships between the species studied. We discuss the possibility that macro- and microchaetes may have different functions and that these have implications for evolutionary constraints on bristle patterns.
The occurrence of complex coacervation in an aqueous mixture of proteins (lysozyme, albumin, immunoglobulin G) and hyaluronic acid and its effect on protein transition in a model system was studied ...to elucidate factors determining the bioavailability of subcutaneously injected therapeutic proteins. Mixing of hyaluronic acid and the model proteins induced complex coacervation at solution pH close to or below the isoelectric point of the proteins. In vitro dialysis using membranes with large pore size tube represented a limitation in the protein transition of the coacervation mixture. Thermal analysis suggested there was retention of the protein conformation in the polymer complex.
Achieving appropriate inhalation in patients with coronavirus disease 2019 (COVID-19) is a common challenge in the use of repurposed metered-dose inhaler (MDI) formulations. The purpose of this study ...was to evaluate the effect of five valved holding chambers (VHCs) on the inhalation of ciclesonide from Alvesco MDI. The aerodynamic particle size distribution of ciclesonide discharged from Alvesco MDI was evaluated using a Next Generation Impactor in the presence and absence of VHCs. The use of VHCs retained or slightly increased the amount of ciclesonide in the fine particle diameter range (aerodynamic particle size below 3 μm) (FPD) and reduced the amount at the induction port after coordinated inhalation. However, the use of VHC reduced the FPD of the formulation by increasing the time between the MDI discharge and the pump suction by various degrees among the five VHCs. These results indicated that use of the VHCs and minimizing the inhalation delay time should ensure sufficient inhalation of ciclesonide particles.
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