Takeshi Mori

Single-walled carbon nanotubes (SWCNTs) are promising nanomaterials for cancer imaging and drug delivery owing to their near-infrared optical properties and tumor accumulation via the enhanced permeability and retention effect. Polyethylene glycol-modified phospholipids (PL-PEG) are widely used to disperse SWCNTs for in vivo applications; however, whether this formulation represents the optimal PEGylation strategy, particularly under repeated administration, remains unclear. Here, we perform a systematic head-to-head comparison between conventional 5 kDa PL-PEG/SWCNTs and SWCNTs encapsulated within a PEG-based cross-linked polymer network synthesized via CNT micelle polymerization. Quantitative biodistribution analysis using Raman spectroscopy demonstrates that cross-linked PEG/EAA/SWCNTs exhibit blood circulation profiles and tumor accumulation comparable to those of PL-PEG/SWCNTs following initial intravenous injection in mice. Striking differences emerge upon repeated dosing. PL-PEG/SWCNTs display reduced systemic exposure consistent with accelerated blood clearance (ABC)-like pharmacokinetics, accompanied by a trend of increased hepatic accumulation. In contrast, PEG/EAA/SWCNTs maintain largely preserved pharmacokinetic profiles (ABC index ≈ 0.8–0.9) and induce significantly lower anti-PEG IgM responses. These results suggest that the observed differences are attributable to differences in PEG molecular weight and/or PEG presentation architecture. By expanding the design space beyond the widely used PL-PEG standard, this study identifies PEG/EAA/SWCNTs as a promising alternative formulation for repeated-dose biomedical applications and highlights the importance of systematically evaluating PEG molecular weight and surface presentation in future studies.

Rheumatoid arthritis is characterized by chronic synovial inflammation and recurrent flares, which are thought to involve joint-specific inflammatory memory. Synovial fibroblasts (SFs) contribute to this process through altered responses to repeated inflammatory stimulation. However, pharmacological targeting of these state-dependent responses remains limited, partly due to the lack of screening systems that distinguish responses under initial and repeated stimulation conditions. Here, we established a quantitative in vitro system using primary murine SFs. Repeated tumor necrosis factor-α stimulation enhanced interleukin-6 (IL-6) production, recapitulating key features previously reported in human SFs. This system enabled direct comparison of drug effects under single and repeated stimulation conditions, revealing distinct response patterns and identifying compounds that selectively modulate the enhanced response induced by repeated stimulation. Notably, a complement receptor antagonist attenuated the additional increase in IL-6 production observed after repeated stimulation, accompanied by the attenuation of glycolysis-related gene expression, while showing no significant effect under single stimulation conditions. These findings highlight the importance of state-dependent pharmacological evaluation and demonstrate the utility of this experimental framework for analyzing differential responses associated with repeated inflammatory stimulation.

Acidic-environment targeting peptides (AEPs), which insert into cell membranes under acidic conditions, have been gaining attention as potential targeting ligands for acidic tissues such as tumors. Conventional AEPs have been taken from archaea or designed rationally; therefore, they have a risk of antigenicity. Here, we propose screening for AEPs from the human proteome. AEP candidates were screened from more than 20000 human proteome transmembrane peptides based on 4 conditions AEPs should fulfill. Twenty-seven peptides were found to satisfy the four conditions. The same number of candidate peptides were identified in the mouse membrane proteome, most of which originated from the orthologous membrane proteins identified in the human proteome. Four of the 27 peptides selected from the human proteome were synthesized with a fluorescence label attached or expressed as fusion proteins with green fluorescent protein to examine their acid-responsive accumulation in vitro and in vivo. We found that 1 of the 4 peptides performed similarly to the pH-low insertion peptide, the first reported AEP.

Poly(ethylene glycol) (PEG) is widely used as a stealth polymer to enhance drug stability and circulation by reducing immune recognition. However, anti-PEG antibodies are increasingly reported in humans, leading to accelerated drug clearance and adverse immune responses. While ensemble assays have clarified the scheme of PEG-antibody binding, they lack the resolution to probe molecular-scale mechanics. Here, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to examine how PEG terminal chemistry and antibody maturation modulate these interactions. Methoxy- (m-PEG) and hydroxy-terminated PEG (HO-PEG) were tested against Fv-clasps from two anti-PEG IgMs: the naïve IgM M9 and the affinity-matured IgM M11. M11 bound PEG more strongly and at shorter rupture distances than M9, with 2D force-distance maps revealing the most intense signatures for M11 and m-PEG pair. Complementary quartz crystal microbalance with dissipation (QCM-D) and Fourier-transform infrared (FTIR) spectroscopy confirmed higher binding by M11 and a terminal preference of M9 for m-PEG. In addition to antibody maturation, we report that the hydrated structure of PEG plays a significant role in PEG-antibody binding. HO-PEG forms extended, hydrated layers, whereas m-PEG adopts compact, collapsed conformations, shaping antibody accessibility and binding mechanics. These results provide molecular-level insight into how antibody structure and PEG hydration state dictate binding, offering design principles for PEGylated therapeutics with reduced immunogenicity and improved performance.

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) fabricated using a water-in-oil-in-water (w/o/w) double emulsion method can simultaneously encapsulate both hydrophilic proteins and hydrophobic drugs. This dual-loading ability of PLGA NPs is expected to be useful in allergen immunotherapy (AIT). We used the formulation to co-encapsulate a hydrophilic model antigen, ovalbumin (OVA), with hydrophobic drugs including rapamycin (Rapa), suberoylanilide hydroxamic acid (Saha) and bexarotene (Bexa). We successfully prepared NPs of approximately 100[ ]nm in diameter and co-encapsulated OVA and the hydrophobic drugs. The NPs remained in the intestinal tract for up to 4[ ]h following oral administration. We found that the NPs showed minimal release of the contents in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) and maintained stability without aggregation or degradation for 8[ ]h. These findings suggest that the NPs have the potential to serve as oral carriers for various hydrophobic drugs and antigens, while maintaining stability in gastrointestinal (GI) environments.

Antisense oligonucleotides (ASOs) were recently approved for treating diseases that are unable to be treated by conventional modalities. However, the approved ASOs target limited organs, particularly the liver, as a result of their pharmacokinetic nature. Here, we report a detailed analysis of the biodistribution and gene knockdown (KD) activity of ASO in organs in the whole body by using ASO incorporated into nanoparticles (NPs). Biodegradable poly(lactide-co-glycolic acid) was used as a matrix of NPs and the Malat1 gene, which is expressed in many organs, was selected as a target of ASO. NPs enabled dramatically high accumulation of ASO in the liver, spleen, lung, and heart compared with that of naked ASO in mice. Notably, high accumulation in the organ did not always result in the KD effect because of the difference of susceptibility to the KD effect in cells that take up NPs. While we used 1 order of magnitude lower dose of ASO compared with conventional naked ASO treatments, NPs showed KD effects not only in the heart but also in the lung and kidney, where naked ASO did not show KD effects. Here, we clarify the unique biodistribution and KD effects of ASO incorporated in NPs. These findings will contribute to the development of an ASO delivery system to target atypical organs with reduced side effects.

This study explores the development of thermosensitive nanostructured coacervates formed by mixing ABA and AB block copolymers. Coacervates are liquid–liquid phase separation systems that can concentrate specific biomolecules, making them useful for biomedical applications such as sustained drug release. This study focuses on creating complex coacervates using ABA-type triblock copolymers that can bridge isolated polyion complex (PIC) domains, enabling the formation of well-ordered assemblies of polyethylene glycol (PEG)-conjugated PIC nanoparticles. These coacervates exhibit reversible responses to temperature changes and morphological hysteresis upon cooling, allowing for the controlled release of PIC micelles at physiological temperatures in the zero-order kinetics. This study demonstrates that the degree of bridging of PIC domains can be tuned simply by adjusting the blend ratio of diblock to triblock copolymers, which affects their physical properties and responsiveness. Real-time and snapshot observations of the assembling behaviors upon heating clarify the formation process of coacervates from both nano- and microscale viewpoints. The successful sustained release of a model protein, green fluorescent protein, is also confirmed to occur in a zero-order manner. These findings suggest that these coacervates are promising depot formulations for nanomedicine, offering a novel model for studying intracellular biomolecular condensates and an intervention method for condensates.

Purpose: Identification and quantification of sulfide ion in biological samples are required in forensic purpose. Gas chromatography-mass spectrometry (GC/MS) has been used for the analysis of sulfide ion by using derivatization reagents. However, conventional derivatization reagents require special attention for derivatization. To simplify the derivatization protocol, we examined ethenesulfonyl fluoride (ESF) as a derivatizing reagent of sulfide ion. Methods: To 100 μL of whole blood sample containing sulfide ion, 100 μL of boric acid buffer (pH 8.0), 100 μL of acetone solution containing internal standard, 100 μL of acetone solution containing 600 mM concentration of ESF, and 100 μL of hexane were added in a 1.5-mL plastic tube. The mixture was vortexed at room temperature, the tubes were centrifuged, and the organic layer was injected into the GC/MS. Results: ESF exhibited higher reactivity toward sulfide ion than interfering compounds present in whole blood, allowing for selective derivatization. With the optimized protocol, the detection limit for sulfide ion was 0.01 μg/mL. The calibration curve showed good linearity (R ² = 0.9999) in the range of 0.05–10.0 μg/mL, and the precision (% relative standard deviation) and the accuracy (% bias) were within ± 10% (intra- and inter-day). Conclusion: This GC/MS-based method is a valuable tool for forensic investigations and various analytical fields, offering reliable quantification of sulfide ion in whole blood.