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Thermo-responsive targeting of polymeric micelles by controlling the cellular uptake based on the change of their surface arginine density.

Yamada S., Sasaki E., Ohno H., Nagase K., Hanaoka K.

Communications Chemistry 8 ( 1 ) 325 2025.11

Research paper (scientific journal), Lead author, Accepted, ISSN 23993669

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Stimulus-responsive nanocarriers are good candidates for targeted drug delivery. Herein, inspired by the existence of a clear threshold number of arginine residues in oligoarginines for cell-penetrating peptide (CPP) activity, we developed a strategy to control the CPP activity by changing the local arginine density for thermo-responsive targeting. We constructed polymeric micelles whose shell consists of a thermo-responsive polymer based on N-isopropylacrylamide, with a low density of arginine moieties (named Arg-TRM). At physiological temperature (37 °C), internalization of Arg-TRM into cells was small and comparable to that of micelle without arginine. In contrast, upon heating at 42 °C, the arginine density on the micellar surface was increased by thermo-responsive shrinkage of the shell, thereby switching on the CPP activity and enabling efficient cellular uptake. The response of Arg-TRM at 42 °C occurred within a few minutes and the intracellular uptake was rapidly enhanced from 5 min after the heating. This response was transient, thus enabling reversible control of the enhancement by heating. As proof-of-concept, we show that intravenously administered Arg-TRM was effectively accumulated in one ear of a normal mouse by local heating. These results indicate that Arg-TRM is a promising drug carrier for on-demand targeted drug delivery in response to mild external heating.
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Heat-guided drug delivery via thermally induced crosslinking of polymeric micelles

Yamada S., Sasaki E., Ohno H., Hanaoka K.

Communications Chemistry 7 ( 1 ) 287 2024.12

Research paper (scientific journal), Lead author, Accepted, ISSN 23993669

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Targeted drug delivery in response to external stimuli is therapeutically desirable, but long-term drug retention at the target site after stimulation is turned off remains a challenge. Herein, we present a targeted-delivery strategy via irreversible aggregation of drug carriers in response to mild external heating. We constructed two types of polymeric micelles, DBCO-TRM and Az-TRM, having a thermo-responsive polymer shell based on N-isopropylacrylamide (NIPAAm) and incorporating alkyne and azide moieties, respectively. Upon heating at 42 °C, the micelles aggregated through hydrophobic interaction between their dehydrated shells. Further, the azide moieties of Az-TRM become exposed on the surface due to the thermally shrinkage of the shells, thereby enabling crosslinking between the two types of micelles via azide-alkyne click chemistry to form irreversible aggregates. These aggregates were efficiently accumulated at tumor sites in mice by local heating after intravenous administration of a mixture of the micelles, and were well retained after cessation of heating due to their increased size. As proof of concept, we show that delivery of doxorubicin in this heat-guided drug delivery system dramatically improved the anti-tumor effect in a mouse model after a single treatment. Our results suggest that this platform could be an efficient tool for on-demand drug delivery.
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Enzymes that generate and regulate intracellular persulfides and polysulfides: mechanistic insights and inhibitors

Hirabayashi K., Sasaki E., Ohno H., Takayama O., Yamada S., Hanaoka K.

Frontiers in Physiology 17 1764165 2026.02

Accepted

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Reactive sulfur species (RSS), which include various persulfides and polysulfides, are generated by multiple enzymes in vivo and play critical roles in mammalian physiological processes such as redox signaling, metabolic regulation, radical scavenging and anti-inflammatory responses. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) are well known to mediate endogenous production of hydrogen sulfide (H<inf>2</inf>S), and, together with the mitochondrial isoform of cysteinyl-tRNA synthetase (CARS2), are proposed to be major sources of intracellular persulfides and polysulfides. In mitochondria, enzymes involved in the sulfide oxidation pathway, including sulfide:quinone oxidoreductase (SQOR), persulfide dioxygenase (ETHE1) and thiosulfate sulfurtransferase (TST), also contribute to maintaining and regulating intracellular persulfide levels. Selective inhibitors targeting these enzymes are expected to be powerful tools for elucidating the functions of RSS, as well as having therapeutic potential. In this review, we present a comprehensive overview of these enzymes, focusing on their reaction mechanisms and inhibitors.
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Development of a silicon phthalocyanine analogue for near-infrared photoimmunotherapy and its application to HTLV-1-infected leukemic cells

Fuse Y., Sasaki E., Tamaki M., Kawamura S., Ohno H., Yamada S., Yasunaga M., Takakura H., Hanaoka H., Kobayashi H., Nakasone H., Hanaoka K.

Rsc Chemical Biology 6 ( 10 ) 1576 - 1584 2025.10

Research paper (scientific journal), Accepted

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Near-infrared photoimmunotherapy (NIR-PIT) employing an antibody labeled with a silicon phthalocyanine dye, IR700, was approved as a minimally invasive treatment for unresectable recurrent head and neck cancer in Japan in 2020. However, further derivatization of IR700 is needed to increase the efficiency of cancer treatment. Here, we developed SiPc-1 as an IR700 analog, in which the linker was constructed using click chemistry to simplify the synthetic scheme and its position was switched from α to β on the benzene ring of phthalocyanine to eliminate intramolecular steric repulsion. We evaluated the cleavage rate of the water-soluble axial moieties of SiPc-1 upon photoirradiation, the cytotoxicity, and the morphological change (blebbing) of treated cells upon photoirradiation. We performed gene expression and protein expression analyses to find a target antigen selectively expressed on cells infected with human T-cell lymphotropic virus type 1 (HTLV-1), the causative virus of adult T-cell leukemia/lymphoma (ATL), and identified CD25 as a suitable target antigen. An anti-CD25 antibody, basiliximab, labeled with SiPc-1 (bas-SiPc-1) showed selective toxicity towards HTLV-1-infected cultured cells and ATL patients’ peripheral blood mononuclear cells upon photoirradiation.
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Trans-omics landscape of systemic vasculitis identified matrix metalloproteinase 12 as a novel signature molecule

Matsumoto K., Suzuki K., Magi M., Onishi S., Yoshida H., Takeshita M., Kuramoto J., Yazawa M., Kato T., Shimizu H., Ito T., Yamada S., Sasaki E., Hanaoka K., Noguchi-Sasaki M., Matsumoto Y., Takeuchi T., Kaneko Y.

Rheumatology 64 ( 8 ) 4766 - 4775 2025.08

Research paper (scientific journal), Accepted, ISSN 14620324

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Objectives To identify key molecules involved in the disease pathophysiology of systemic vasculitis through trans-omics analysis, with a specific focus on demonstrating matrix metalloproteinase (MMP) 12 as a potential biomarker in rheumatic entities. Methods Patients with newly diagnosed or relapsed rheumatic and musculoskeletal diseases from June 2013 until September 2022 were enrolled. We screened vasculitis-specific molecules by combining findings from serum proteome analysis and whole-blood RNA sequencing. We further validated the identified molecules using immunohistochemical staining and spatial transcriptome analysis. Results Serum proteome and RNA sequencing identified MMP12 as a significant molecule for systemic vasculitis; it distinguished vasculitis from other rheumatic and musculoskeletal diseases, reflected disease activity along with longitudinal change, and predicted relapse in patients with large-vessel vasculitis. MMP12 could also be used to detect insidious disease activity even under treatment with IL-6 inhibition. Immunohistochemical staining of the affected tissues demonstrated that MMP12 was specifically expressed in tissue-infiltrating CD206-positive macrophages. Spatial transcriptome analysis revealed the characteristic phenotype of MMP12-positive macrophages and its association with macrophage maturation and formation of multinucleated giant cells. Conclusion MMP12 is a disease-specific molecule that is associated with macrophage maturation and the formation of multinucleated giant cells and reflects disease activity independently of the IL-6 pathway in systemic vasculitis.
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Enzymes that generate and regulate intracellular persulfides and polysulfides: mechanistic insights and inhibitors

Hirabayashi K., Sasaki E., Ohno H., Takayama O., Yamada S., Hanaoka K.

Frontiers in Physiology 17 1764165 2026.02

Article, review, commentary, editorial, etc. (scientific journal)

　View Summary

Reactive sulfur species (RSS), which include various persulfides and polysulfides, are generated by multiple enzymes in vivo and play critical roles in mammalian physiological processes such as redox signaling, metabolic regulation, radical scavenging and anti-inflammatory responses. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) are well known to mediate endogenous production of hydrogen sulfide (H2S), and, together with the mitochondrial isoform of cysteinyl-tRNA synthetase (CARS2), are proposed to be major sources of intracellular persulfides and polysulfides. In mitochondria, enzymes involved in the sulfide oxidation pathway, including sulfide:quinone oxidoreductase (SQOR), persulfide dioxygenase (ETHE1) and thiosulfate sulfurtransferase (TST), also contribute to maintaining and regulating intracellular persulfide levels. Selective inhibitors targeting these enzymes are expected to be powerful tools for elucidating the functions of RSS, as well as having therapeutic potential. In this review, we present a comprehensive overview of these enzymes, focusing on their reaction mechanisms and inhibitors.
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Recent advances in fluorogenic probes based on twisted intramolecular charge transfer (TICT) for live-cell imaging

Ohno H., Sumitani S., Sasaki E., Yamada S., Hanaoka K.

Chemical Communications 61 ( 69 ) 12871 - 12884 2025.08

Article, review, commentary, editorial, etc. (scientific journal), ISSN 13597345

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Fluorescence imaging is a powerful technique for visualizing biological events in living samples, and new fluorescence-control mechanisms are still needed to extend the scope of biomolecule-targeting fluorogenic probes. Twisted intramolecular charge transfer (TICT) is a unique fluorescence quenching mechanism that depends upon a twisted conformation to promote intramolecular charge separation. Probes utilizing TICT can detect biological molecules/phenomena, such as viscosity, polarity and extended protein structures, that cannot readily be accessed by probes employing other fluorescence-control mechanisms, such as photoinduced electron transfer or spirocyclization. In this review, we summarize recent work on molecular design strategies for TICT-based fluorogenic probes, focusing on structural-modification approaches to control the ease of TICT state formation.
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アルギニン密度の温度スイッチングによる細胞内取込制御

山田創太，花岡健二郎

ケミカルバイオロジー 18 ( 1 ) 10 - 12 2025.06

Article, review, commentary, editorial, etc. (scientific journal), Lead author
熱に応答した架橋形成によるナノ粒子の標的選択的送達

山田創太，花岡健二郎

JSMI Report 18 ( 1 ) 31 - 35 2025.02

Article, review, commentary, editorial, etc. (scientific journal), Lead author
Recent advances in Si-rhodamine-based fluorescent probes for live-cell imaging

Ohno H., Sasaki E., Yamada S., Hanaoka K.

Organic and Biomolecular Chemistry 22 ( 16 ) 3099 - 3108 2024.03

Article, review, commentary, editorial, etc. (scientific journal), ISSN 14770520

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Fluorescence imaging is a powerful technique for visualizing biological events in living samples with high temporal and spatial resolution. Fluorescent probes emitting far-red to near infrared (NIR) fluorescence are particularly advantageous for in vivo imaging due to their high tissue permeability and low autofluorescence, as well as their suitability for multicolor imaging. Among the far-red to NIR fluorophores, Si-rhodamine is one of the most practical fluorophores for the development of tailor-made NIR fluorescent probes because of the relative ease of synthesis of various derivatives, the unique intramolecular spirocyclization behavior, and the relatively high water solubility and high photostability of the probes. This review summarizes these features of Si-rhodamines and presents recent advances in the synthesis and applications of far-red to NIR fluorescent probes based on Si-rhodamines, focusing on live-cell imaging applications such as fluorogenic probes, super-resolution imaging and dye-protein hybrid-based indicators.
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