Yoshiki Katayama

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.

We previously reported the development of inflammation-triggering engineered mouse macrophages (mMacTriggers) that achieve tumor-specific release of tumor necrosis factor-α (TNF-α) in response to the promoter of Arginase 1 (Arg1), an M2-specific marker in murine macrophages. Tumor-specific TNF-α release induced acute inflammation, which recruited natural killer cells or cytotoxic T cells to attack the tumor, leading to significant antitumor effects. This strategy primarily leverages innate immunity-mediated tumor suppression, which is expected to have minimal side effects—an approach noted worldwide. However, we have one issue with preparing human macrophage-derived MacTrigger (hMacTrigger) for clinical application: the Arg1 promoter is unsuitable for human use due to the low expression of Arg1 in human macrophages. Therefore, this study aimed to identify human M2 macrophage-specific markers to replace murine Arg1 through data analysis and quantitative evaluation. From an initial selection of 30 gene candidates by data analysis, we identified 8 genes with high specificity by quantitative evaluation. Among them, 4 genes—arachidonate 15-lipoxygenase (ALOX15), sialic acid-binding immunoglobulin (Ig)-like lectin 10 (SIGLEC10), fatty acid binding protein 4 (FABP4), and C-C motif chemokine ligand 22 (CCL22)—were confirmed as M2-specific markers in human macrophages. Future research will focus on preparing hMacTrigger by constructing vectors encoding TNF-α under the control of each identified gene promoter and evaluating its anti-tumor effects and side effects in vivo. This study represents a critical step toward the clinical application of the MacTrigger strategy and advances its potential use in cancer immunotherapy.

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.

Background/Aim: Chimeric antigen receptor T-cell therapy has shown efficacy against chemoresistant B-cell leukemia and lymphoma but is limited in solid tumors. This study proposes using inflammation-triggering engineered macrophages (MacTriggers) to target chemoresistant tumors. Intravenous MacTriggers infiltrate tumors, inducing inflammation via tumor necrosis factor-alpha (TNF-α), converting the immunosuppressive microenvironment into an immuno-active state, and enhancing anti-tumor immune responses. Materials and Methods: DOX-resistant murine colon cancer cells (DOX-Resi) were established by repeated in vivo exposure to DOX. IC₅₀ values and mRNA expression of Abcb1a (encoding P-gp) in WT or DOX-Resi cells were evaluated by qPCR. MacTriggers were engineered to release TNF-α upon sensing tumor-associated arginase 1 (Arg1) activity. BALB/c mice with subcutaneous DOX-Resi tumors received intravenous MacTriggers or DOX. Tumor growth, histological changes, and side effects, including cardiotoxicity, were assessed via tumor volume monitoring, immunohistochemistry, and serum cardiac troponin-I measurement. Results: DOX-Resi cells had an IC₅₀ value approximately 2.5 times higher than WT cells, with significantly higher Abcb1a expression. MacTriggers significantly suppressed DOX-Resi tumor growth, while DOX showed limited efficacy. MacTrigger administration did not cause severe side effects, unlike DOX, which induced cardiotoxicity. Conclusion: MacTriggers offer a novel, effective, and safer therapeutic approach for chemoresistant solid tumors, addressing chemotherapy limitations and improving outcomes in drug-resistant cancers.

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.

We proposed a novel ligand for the interaction with human serum albumin (HSA) to extend the blood half-life of small molecular weight therapeutics. The ligand features an alkyl chain and an activated disulfide to allow binding to the hydrophobic pockets of HSA and the formation of disulfide to Cys34 of HSA, thereby minimizing the initial renal clearance. The dual nature of the ligand-HSA bonding was expected to give the ligand long blood retention. After 1 min of mixing with HSA, the ligand showed higher binding (1.7 times) than that of a control ligand (containing only activated disulfide). After intravenous injection to mice, the ligand half-lives were 1.6 and 9.2 times longer than those of control ligands with the active disulfide alone and with the alkyl chain alone, respectively. The proposed ligand has the potential to act as a platform for extending the half-life of small therapeutics in vivo.

Background: We have previously reported engineered macrophages (MacTriggers) that can accelerate the release of tumor necrosis factor-α in response to M2 polarization. MacTriggers are characterized by two original characteristics of macrophages: (1) migration to tumors; and (2) polarization to the M2 phenotype in tumors. Intravenously administered MacTriggers efficiently accumulated in the tumors and induced tumor-specific inflammation. This study reports a novel methodology for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Results: In this study, we newly found that the intravenously administered MacTriggers in BALB/c mouse models upregulated the expression levels of immune checkpoint proteins, such as programmed cell death (PD)-1 in CD8⁺ T cells and PD-ligand 1 (PD-L1) in cancer cells and macrophages. Consequently, in two ICI-resistant tumor-inoculated mouse models, the combined administration of MacTrigger and anti-PD-1 antibody (aPD-1) synergistically inhibited tumor growth, whereas monotherapy with aPD-1 did not exhibit anti-tumor effects. This synergistic effect was mainly from aPD-1 enhancing the tumor-attacking ability of CD8⁺ T cells, which could infiltrate into the tumors following MacTrigger treatment. Importantly, no side effects were observed in normal tissues, particularly in the liver and spleen, indicating that the MacTriggers did not enhance the aPD-1 reactivity in normal tissues. This specificity was from the MacTriggers not polarizing to the M2 phenotype in normal tissues, thereby avoiding inflammation and increased PD-1/PD-L1 expression. MacTriggers could enhance aPD-1 reactivity only in tumors following tumor-specific inflammation induction. Conclusions: Our findings suggest that the MacTrigger and aPD-1 combination therapy is a novel approach for potentially overcoming the current low ICI response rates while avoiding side effects.

Background: Acrylamide polymers with zwitterionic carboxybetaine (CB) side groups have attracted attention as stealth polymers that do not induce antibodies when conjugated to proteins. However, they induce antibodies when modified onto liposomes. We hypothesized that antibodies are produced against polymer backbones rather than CB side groups. Objectives: In this study, we designed and synthesized a polymer employing CB in its main chain, poly(N-acetic acid-N-methyl-propyleneimine) (PAMPI), and evaluated the blood retention of PAMPI-modified liposomes in mice. Results: The non-fouling nature of PAMPI-modified liposomes estimated from serum protein adsorption was found to be not inferior to PCB- and PEG-modified liposomes. However, to our surprise, the PAMPI-modified liposomes showed an instantaneous clearance less than 1 h post-injection, comparable to the naked liposomes. Conclusions: The extent of the blood retention of polymer-modified liposomes cannot be predicted by their susceptibility to serum protein adsorption and semi-flexible conformation.

This study investigates the impact of hydrophobic modification on the immunogenicity, cytotoxicity, and inflammatory response of Alaska pollock gelatin (ApGltn) microparticles (MPs). Gelatin, known for its inherent biocompatibility, was modified with decyl group (C10) to explore potential alterations in its interaction with the immune system. Immunogenicity was evaluated through the measurement of material-specific IgM and IgG responses, indicating no significant increase post-modification. Cytotoxicity against Caco-2 cell lines and NF-κB-mediated LPS-induced inflammation were also assessed, revealing no exacerbation by the modified MPs. Furthermore, C10 modification with different types of linkage such as secondary amine and amide structure did not influence immune reactivity. These findings suggest that C10 modification maintains the non-immunogenicity and biocompatibility of gelatin MPs, supporting their potential use in biomedical applications. Graphical abstract