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.

Achieving antigen-specific immune tolerance without systemic immunosuppression remains a major challenge in biomaterial-based immunotherapy. Here, we report a simple nanoparticle (NP)-based platform that enables a transient, B cell-targeted tolerance switch. NPs encapsulating metabolizable aryl hydrocarbon receptor (AhR) agonists-FICZ or ITE-preferentially accumulate in splenic marginal zone B cells and convert them into IL-10-producing regulatory B cells (Bregs). This study provides the first in vivo evidence that Bregs can directly present antigen and induce regulatory T cells (Tregs), establishing a NP-controlled Breg-Treg pathway. These Bregs promote antigen-specific Tregs expansion only when co-exposed to antigen, establishing time-gated, antigen-restricted immune regulation. By exploiting the rapid metabolism of AhR agonists, this system provides precise temporal control of tolerance induction while preserving vaccine responses. In mouse models, co-administration of FICZ-containing NP with antigen suppressed anti-drug antibody formation and ameliorated allergic inflammation. This NP platform demonstrates a strategy for safe, antigen-specific immunomodulation and offers a clinically adaptable framework for allergy and biotherapeutic tolerance.

This study explores 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 mostly 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.

Effective siRNA therapy requires efficient delivery systems. However, sustained-release technologies remain poorly developed. A novel coacervate-based formulation that enables the controlled release of siRNA-loaded polyion complex micelles was developed in this study. An ABA-type triblock copolymer (catiomer–polyethylene glycol–catiomer) and an AB-type diblock copolymer were blended at defined ratios (fTri) and complexed with siRNA/aniomer mixtures (fsiRNA) to form polyion complex assemblies. Certain formulations generated micelles at ambient temperature, which thermally transitioned into coacervated microparticles (transition temperature: 45 °C at fTri = fsiRNA = 0.4) and reverted to micelles upon cooling. At 37 °C, the coacervates released siRNA and micelles at a constant rate. Cytotoxicity was negligible after 24 h but increased after 72 h. Early-stage uptake (≤3 d), assessed using flow cytometry and confocal microscopy, revealed superior internalization for coacervates versus micelles, primarily via small PIC species, considering the release behavior of the initial stage. Luciferase knockdown assays confirmed enhanced gene silencing for the coacervate-treatment groups, as compared to the micelle-treated groups, although the overall efficiency remained modest. These findings demonstrate that coacervate-based formulations enable sustained siRNA release and improved cellular uptake, as compared with micelles, highlighting their potential as next-generation delivery systems. A thermoresponsive coacervate enabling the sustained release of micelles and siRNA was developed using an appropriate blend of ABA/AB block copolymers. At room temperature, siRNA-loaded polyion complex (PIC) micelles were dispersed in solution. Upon mild heating ( ≈ 45 °C), they assembled into coacervate microparticles through a reversible phase transition. When maintained at 37 °C, the coacervates continuously and constantly release siRNA and micelles. Biological activities were evaluated using living cells, and cellular uptake and gene knockdown effects were confirmed. This concept highlights a coacervate-based depot platform for siRNA delivery system.

Liquid–liquid phase separation leads to the formation of liquid droplets (LqDs) such as P granules in Caenorhabditis elegans (C. elegans). In this study, we demonstrate the label-free visualization of LqDs using multimodal nonlinear optical imaging both in vitro and in vivo. In vitro measurements with polymerized adenine [poly(A)], we found significantly higher poly(A) concentrations in LqDs compared to surrounding solutions, with the limit of detection (LoD) of 32 mg/mL. In vivo measurements, we performed label-free imaging of C. elegans. Despite efforts to detect P granules within P lineage cells in both wild-type C. elegans and green fluorescent protein (GFP)-tagged strains, no clear RNA-specific signals were observed. This indicates that the RNA concentration in P granules is lower than anticipated and falls below our in vitro LoD. These results underscore the challenges of label-free RNA detection in P granules.

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 100nm in diameter and co-encapsulated OVA and the hydrophobic drugs. The NPs remained in the intestinal tract for up to 4h 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 8h. 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.

Biomolecular condensates offer a versatile platform for the accumulation of biomacromolecules, particularly proteins. This study investigated synthetic complex coacervates as a model of biomolecular condensates and developed a novel tool for the intracellular delivery of proteins to overcome the issues of cytotoxicity and poor cellular uptake. By optimizing preparation conditions and chemical structures and developing a simple pre-coating method, we achieved an improvement in the interaction and internalization of coacervates for HeLa and Jurkat cells and the reduction of cytotoxicity. Furthermore, the use of charge-density reduced polymers enabled the effective encapsulation and intracellular delivery of various proteins. The mechanism of cellular internalization was also investigated, and macropinocytosis was concluded to be a primary internalization pathway. These findings provide a foundation for future advancements in biomaterials.

As the potential applications of nanoparticles (NPs) in insect pest management continue to be explored, the focus has primarily been on external feeders, leaving a notable knowledge gap regarding internal leaf feeders. In this study, we investigated the effects of silica (SiO2), titania (TiO2), and silver (Ag) NPs on the American serpentine leafminer, Liriomyza trifolii (Diptera: Agromyzidae), a devastating pest of a diverse array of crops. NPs were sprayed on the leaves of seedlings of the common bean, Phaseolus vulgaris (Fabaceae), at concentrations of 50, 100, 200, and 400 mg/L to evaluate their effects on the survival, development, feeding rate, and body mass/size of the leafminer. qRT-PCR was used to assess oxidative stress in pupae based on the expression of genes for two major antioxidant enzymes, catalase (CAT) and superoxide dismutase 2 (SOD2). Total protein content was also quantified. Compared to the control (distilled water), neither SiO2, TiO2, nor Ag NPs affected larval feeding rate. SiO2NPs decreased puparia weight, while TiO2 and Ag NPs increased both the weight and length of puparium as well as wing length in adults. The lowest tested concentration of TiO2NPs (50 mg/L) and the highest of AgNPs (400 mg/L) led to upregulation of SOD2, whereas SiO2NPs had no significant effects on the expression of either CAT or SOD2. Total protein content was not affected by any NP treatment. Further assessment of the effects of SiO2NPs revealed fewer observed mines, reduced larval survival, leading to a reduction in the number of emerging adults at 400 mg/L, delay in mine appearance, pupariation, and adult emergence at varying concentrations, and visible wing deformities; the LC50 of SiO2NPs was estimated to be 550 mg/L. Ultimately, because the preapplication of TiO2 and Ag NPs on P. vulgaris increased the body size (wing length) of emerging adult leafminers, compared to the largely negative effects of SiO2NPs, plant-mediated chronic exposure to TiO2 and Ag NPs at sublethal concentrations may increase rather than decrease the performance of phytophagous insects. Our results have important implications for the use of NPs in the management of internal feeders and other insect pests.

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.

Inhibitors of the calciprotein particle (CPP) maturation have been developed so far as therapeutics for vascular calcification. However, the short blood half-life limited their application. Here we designed the conjugate of a CPP maturation inhibitor, alendronate (ALN) with human serum albumin (HSA) to utilize the long blood retention nature of HSA. The HSA-ALN conjugates with different modification numbers of ALN per HSA were prepared. The inhibitory effect of the conjugates on the CPP maturation was evaluated using a reported cell-free system as a time of conversion from CPPI to CPPII. The CPP binding of fluorescent-labeled conjugates was carried out using flow cytometry. The plasma half-life of the conjugates was evaluated in mice after intravenous injection. We found that the HSA-ALN conjugates bound to CPP via the specific interaction between ALN and calcium phosphate of CPP. As a result, the conjugates showed a much higher inhibition effect of CPP maturation than those of free ALN and intact HSA. A modification ratio of two ALN molecules per HSA was found to be significant enough to inhibit the CPP maturation. Such a small modification ratio minimized the impact on the long blood retention nature of HSA. This study showed that HSA-ALN conjugates not only have superior CPP growth inhibition effects but also possess significantly higher blood half-lives compared to those of free ALN. These findings suggest that HSA-ALN conjugates are promising therapeutics for vascular calcification associated with the deposition of CPP.

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.

Polyion complex vesicles (PICsomes) possess several features that render them as suitable for drug delivery systems. However, retaining water-soluble low-molecular-weight compounds (WLMWCs) remains challenging because of the high permeability of their vesicular membranes. Herein, we propose a new approach for prolonged retention and sustained release of WLMWCs from PICsomes by loading hydroxypropyl methylcellulose to increase the viscosity of the inner aqueous phase. The PICsomes retained 2% to 4% of the WLMWCs, and 100% of the WLMWCs were released within 96 h according to first-order kinetics. Conventional PICsomes cannot retain water-soluble low-molecular-weight compounds (WLMWCs) because the PICsome membrane is highly permeable to them. In this study, utilizing stepwise crosslinking of the membrane, a new method was developed for loading viscosity-enhancing polymers. This new method allowed the encapsulation of hydroxypropyl methylcelluloses, which enabled PICsomes to retain and sustainably release WLMWCs.

This study introduces the α-rhamnose (Rham)-conjugated prodrug of SN-38 (Rham-SN-38) as a promising alternative to irinotecan. α-rhamnosidase, responsible for SN-38 release from Rham-SN-38, does not express in human cells, minimizing individual variability and side effects. The injection of the α-rhamnosidase into the tumor tissues makes it possible, for the first time, to activate the Rham-SN-38. Furthermore, α-rhamnosidase demonstrates significantly higher activity than carboxylesterase, the specific enzyme activating irinotecan. SN-38 release mediated by α-rhamnosidase completes within 2 h, with a kcat/Km value approximately 5.0 × 104-fold higher than that of irinotecan. The 50

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.

Human orthogonal enzymes (HOEs) do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluate a new HOE from Escherichia coli, , alpha-sulfoquinovosidase (alpha-SQase). We confirmed that the activity of alpha-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for alpha-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed a similar reactivity to the p- nitrophenol substrate, a good substrate for alpha-SQase.

Previously, we reported anticancer molecules, Fc-binding antibody-recruiting molecules (Fc-ARMs), which crosslink proteins on cancer cells with endogenous immunoglobulin Gs (IgGs) via their Fc region. The mobilized IgGs on cancer cells can accommodate natural killer cells to induce antibody-dependent cellular cytotoxicity (ADCC). Because previous Fc-ARMs utilized Fc-binding peptides, their affinity to IgGs is weak, which resulted in the limited induction capability of ADCC. Previous Fc-ARMs also unitized small molecular ligands to cancer cells, which limited their universal applicability to any cancer cells. A recent study reported that protein-based Fc-ARMs might overcome the issues associated with non-proteinous Fc-ARMs. Here, we examined the universality of a protein-based Fc-ARM by replacing its tumor-binding domain with a human epidermal growth factor receptor 2 (HER2)-specific affibody (ZHER2:342). We also examined the requirement of its Fc-binding domain affinity. We found that the Fc-ARMs accepted an affibody as a tumor-binding domain to induce ADCC. Furthermore, the required residence time of the complex between Fc-ARM and IgG was ∼102 min, which was comparable to that when monoclonal antibodies bind to their specific antigens. However, we found that the extent of ADCC induced by Fc-ARM was lower than that of conventional IgG-mediated ADCC, indicating that further enhancement of the affinity of the antibody-binding terminus and tumor-binding terminus of Fc-ARM may be needed to achieve ADCC equivalent to that of conventional IgG-mediated ADCC.

Design factors of PEGylated polyion complex (PIC) nanoparticles, particularly PIC vesicles, were examined regarding polyion chain matching, domain crosslinking, and chemical modification of charged units, to modulate mammalian cell-PIC interaction.