Elsevier BV Acta Medica Okayama 2211-7156 25 2026 Peptide nanomicelles for NIR light-dependent siRNA delivery 103265 EN Taufik Fatwa Nur Hakim Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Shoumu Fujimoto Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University The peptide amphiphile PA8, derived from the GAVILRR peptide, was developed as a carrier for small interfering RNA (siRNA) delivery; however, its RNA interference (RNAi) efficacy was limited owing to predominant endocytotic uptake. In this study, the RNAi efficiency of PA8 nanomicelle/siRNA complexes was enhanced by modifying the nanomicelles with the photosensitizer DY750 and the tumor-homing peptide iRGD. The conjugation of DY750 to the nanomicelles facilitated endosomal escape of the nanomicelle/siRNA complexes, enabling the cytosolic release of siRNA. Additionally, the incorporation of iRGD improved RNAi delivery efficiency in the AsPC-1 pancreatic ductal adenocarcinoma cell line. PA8-DY750-iRGD nanomicelle complexes loaded with siRNA against polo-like kinase 1 (PLK1) achieved an 80% reduction in PLK1 mRNA levels in AsPC-1 cells and a moderate 28% knockdown in NCI-N87 gastric cancer cells. Notably, no RNAi effect was observed in noncancerous 1C3D3 pancreatic cells or HEK293T kidney cells, underscoring the selectivity of this system for AsPC-1 cells. These findings highlight the potential of PA8-DY750-iRGD nanomicelle complexes as a targeted therapeutic platform for specific cancers, particularly pancreatic cancer. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 2045-2322 16 1 2026 Liquid–liquid phase separation by caged coacervating peptides 10464 EN Akinari Bando Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Yuuki Kanazaki Department of Applied Chemistry, Kindai University Rika Tojo Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Liquid–liquid phase separation is an important biomolecular process in the formation of membraneless intracellular organelles that has inspired the development of artificial droplet systems. We developed caged coacervating peptides (CCPs) based on a histidine-rich squid beak protein sequence. The peptides were caged with a photodeprotectable (7-diethylaminocoumarin-4-yl)methoxycarbonyl group. The CCPs formed coacervates in the caged state and were partially dispersed upon blue-light irradiation. Photo-uncaging occurred rapidly, inducing coacervate dispersion. A mutant CCP with reduced π–π interactions exhibited efficient photo-dependent disassembly and enabled the encapsulation and release of a fluorescently labeled adenosine 5′-triphosphate (Bodipy-ATP) upon irradiation. These CCPs offer an efficient light-controlled approach for biomolecular encapsulation within coacervates and targeted drug delivery. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 0022-2623 69 5 2026 Discovery of Thermal Sensitizers That Inhibit Heat-Induced SAFB Granule Formation 5944 5955 EN Yuji Furutani Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Natsuki Shimasaki Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Riko Yamada Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Faculty of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hyperthermia is a minimally invasive cancer treatment based on heat stress-induced apoptosis. Its therapeutic efficacy, however, is often limited by tumor heterogeneity and acquired thermotolerance. Therefore, combination strategies involving hyperthermia and chemotherapy have been developed to enhance the therapeutic efficacy. Previously, we showed that SB366791 enhanced heat-induced apoptosis by inhibiting heat stress-induced scaffold attachment factor B (SAFB) granule formation, although its proapoptotic activity was insufficient. Therefore, we screened to identify novel compounds that enhance heat-induced apoptosis by suppressing SAFB granule formation. We identified four hit compounds that inhibited SAFB granule formation, all exhibiting thermal enhancement ratios > 1.0─that significantly enhanced heat-induced apoptosis efficiency. Additionally, the tumor volume in mice treated with a combination of Z19024498 and hyperthermia was significantly smaller than that in mice treated with hyperthermia or Z19024498. These results indicate that the identified compounds, specifically Z19024498, have potential as thermal sensitizers for hyperthermia therapy. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2079-6374 16 1 2026 Magnetic Detection of Cancer Cells Using Tumor-Homing Peptide-Modified Magnetic Nanoparticles 45 EN Shengli Zhou Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yuji Furutani Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kei Yamashita Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sakuya Kako Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshihiko Kiwa Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Magnetic nanoparticles (MNPs) provide a platform for target detection because of their magnetic responsiveness to alternating magnetic fields (AMFs). We developed a detection method using MNPs modified with tumor-homing peptides (THPs), PL1 and PL3, which selectively bind to protein components enriched in malignant tissues. THP-MNPs were synthesized using maleimide-PEG-NHS linkers and characterized using transmission electron microscopy. Human glioblastoma cancer U87MG and normal tissue-derived HEK293 cells were incubated with THP-MNPs, and the magnetic signals were measured using a high-temperature superconducting quantum interference device (SQUID) magnetometer under an AMF (1.06 kHz). Dark-field microscopy confirmed the preferential binding of THP-MNPs to U87MG cells. In the absence of cells, THP-MNPs exhibited AMF-dependent signal enhancement, which correlated with particle size reduction due to THP release. This increase was completely suppressed in the presence of U87MG cells, indicating a strong THP-mediated interaction. PL3-MNPs exhibited superior discrimination between malignant and non-malignant cells. These results demonstrate that SQUID-based magnetic measurements using THP-MNPs enable rapid and label-free cancer cell detection. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 2691-3704 5 2 2025 Mechanistic Insights Into Oxidative Response of Heat Shock Factor 1 Condensates 606 617 EN Soichiro Kawagoe Institute of Advanced Medical Sciences, Tokushima University Motonori Matsusaki Institute of Advanced Medical Sciences, Tokushima University Takuya Mabuchi Frontier Research Institute for Interdisciplinary Sciences, Tohoku University Yuto Ogasawara Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Koichiro Ishimori Department of Chemistry, Faculty of Science, Hokkaido University Tomohide Saio Institute of Advanced Medical Sciences, Tokushima University Heat shock factor 1 (Hsf1), a hub protein in the stress response and cell fate decisions, senses the strength, type, and duration of stress to balance cell survival and death through an unknown mechanism. Recently, changes in the physical property of Hsf1 condensates due to persistent stress have been suggested to trigger apoptosis, highlighting the importance of biological phase separation and transition in cell fate decisions. In this study, the mechanism underlying Hsf1 droplet formation and oxidative response was investigated through 3D refractive index imaging of the internal architecture, corroborated by molecular dynamics simulations and biophysical/biochemical experiments. We found that, in response to oxidative conditions, Hsf1 formed liquid condensates that suppressed its internal mobility. Furthermore, these conditions triggered the hyper-oligomerization of Hsf1, mediated by disulfide bonds and secondary structure stabilization, leading to the formation of dense core particles in the Hsf1 droplet. Collectively, these data demonstrate how the physical property of Hsf1 condensates undergoes an oxidative transition by sensing redox conditions to potentially drive cell fate decisions. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 2772-4174 13 2025 Photochemical internalization of mRNA using a photosensitizer and nucleic acid carriers 100242 EN Hayaki Maemoto Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryohei Suzaki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Keiji Itaka Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University mRNA has great potential for therapeutic applications because it can encode a variety of proteins and antigens, in addition to advantages over DNA in terms of gene expression without genomic integration, nuclear localization, or transcription. However, therapeutic applications of mRNA require safe and effective delivery into target cells. Therefore, we aimed to investigate photochemical internalization (PCI) as a promising strategy for delivering mRNA to target cells. In this strategy, mRNA is taken up into cells by endocytosis, accumulates in endosomes, and is released in a light-dependent manner from the endosomes using an endosome-accumulating photosensitizer, aluminum phthalocyanine disulfonate (AlPcS2a), in combination with nucleic acid carrier molecules. We compared the efficacy of various nucleic acid carriers, including branched polyethyleneimine (bPEI) and poly{N'-[N-(2-aminoethyl)-2-aminoethyl] aspartamide} (PAsp(DET)) under the same conditions for PCI-based mRNA delivery. Our results indicated that bPEI and PAsp(DET) at low N/P ratios exhibited efficient light-enhancement of mRNA expression by PCI with AlPcS2a. Notably, bPEI exhibited the highest light-dependent mRNA delivery among the carriers evaluated (including cationic polymers, cationic peptides, and lipids), whereas PAsp(DET) showed promise for clinical use because of its lower toxicity compared with bPEI. This PCI strategy allows effective cytosolic mRNA delivery at low N/P ratios, thereby reducing cationic carrier molecule-induced cytotoxicity. This method allows spatiotemporal control of protein expression and holds potential for novel light-dependent mRNA therapies. Overall, this study provided valuable insights into optimizing mRNA delivery systems for therapeutic applications. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 14 1 2025 Novel Drug Delivery Particles Can Provide Dual Effects on Cancer "Theranostics" in Boron Neutron Capture Therapy 60 EN Abdul Basith Fithroni Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Haruki Inoue Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shengli Zhou Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Taufik Fatwa Nur Hakim Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Tada Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Minoru Suzuki Institute for Integrated Radiation and Nuclear Science, Kyoto University Yoshinori Sakurai Institute for Integrated Radiation and Nuclear Science, Kyoto University Manabu Ishimoto J-BEAM, Inc. Naoyuki Yamada Nihon Fukushi Fuiin Holding, Co., Ltd. Rani Sauriasari Faculty of Pharmacy, Universitas Indonesia Wolfgang A. G. Sauerwein Deutsche Gesellschaft für Bor-Neutroneneinfangtherapie DGBNCT e.V., University Hospital Essen, Klinik für Strahlentherapie Kazunori Watanabe Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eiji Matsuura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Boron (B) neutron capture therapy (BNCT) is a novel non-invasive targeted cancer therapy based on the nuclear capture reaction 10B (n, alpha) 7Li that enables the death of cancer cells without damaging neighboring normal cells. However, the development of clinically approved boron drugs remains challenging. We have previously reported on self-forming nanoparticles for drug delivery consisting of a biodegradable polymer, namely, “AB-type” Lactosome® nanoparticles (AB-Lac particles)- highly loaded with hydrophobic B compounds, namely o-Carborane (Carb) or 1,2-dihexyl-o-Carborane (diC6-Carb), and the latter (diC6-Carb) especially showed the “molecular glue” effect. Here we present in vivo and ex vivo studies with human pancreatic cancer (AsPC-1) cells to find therapeutically optimal formulas and the appropriate treatment conditions for these particles. The biodistribution of the particles was assessed by the tumor/normal tissue ratio (T/N) in terms of tumor/muscle (T/M) and tumor/blood (T/B) ratios using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG). The in vivo and ex vivo accumulation of B delivered by the injected AB-Lac particles in tumor lesions reached a maximum by 12 h post-injection. Irradiation studies conducted both in vitro and in vivo showed that AB-Lac particles-loaded with either 10B-Carb or 10B-diC6-Carb significantly inhibited the growth of AsPC-1 cancer cells or strongly inhibited their growth, with the latter method being significantly more effective. Surprisingly, a similar in vitro and in vivo irradiation study showed that ICG-labeled AB-Lac particles alone, i.e., without any 10B compounds, also revealed a significant inhibition. Therefore, we expect that our ICG-labeled AB-Lac particles-loaded with 10B compound(s) may be a novel and promising candidate for providing not only NIRF imaging for a practical diagnosis but also the dual therapeutic effects of induced cancer cell death, i.e., “theranostics”. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 29 11 2024 In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia 2632 EN Shengli Zhou Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kaname Tsutsumiuchi College of Bioscience and Biotechnology, Chubu University Ritsuko Imai College of Bioscience and Biotechnology, Chubu University Yukiko Miki College of Bioscience and Biotechnology, Chubu University Anna Kondo College of Bioscience and Biotechnology, Chubu University Hiroshi Nakagawa College of Bioscience and Biotechnology, Chubu University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy. No potential conflict of interest relevant to this article was reported.

nature portfolio Acta Medica Okayama 2045-2322 13 1 2023 Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo 13050 EN Yuka Ikawa Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takuya Wakai Department of Animal Science, Graduate of Environmental and Life Science, Okayama University Hiroaki Funahashi Department of Animal Science, Graduate of Environmental and Life Science, Okayama University Tet Htut Soe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 0003-2654 148 11 2023 FRET probe for detecting two mutations in one EGFR mRNA 2626 2632 EN Myat Thu Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kouta Yanai Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hajime Shigeto Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Shohei Yamamura Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0960-894X 68 2022 Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate 128767 EN Nanako Sumi Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shota Nagahiro Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eiji Nakata Institute of Advanced Energy, Kyoto University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 11 1 2021 Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer 14936 EN Kazunori Watanabe Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tomoko Nawachi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ruriko Okutani Department of Biomedical Engineering, Faculty of Engineering, Okayama University Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Methods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 9 2021 Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress 4982 EN Kazunori Watanabe Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 00223549 110 2021 Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells 1788 1798 EN Melissa Siaw Han Lim Yuki Nishiyama Takashi Ohtsuki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazunori Watanabe Graduate School of Natural Science and Technology, Okayama University Hirotsugu Kobuchi Kazuko Kobayashi Collaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eiji Matsuura The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1420-3049 26 1 2020 Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates 36 EN Tet Htut Soe Department of Biotechnology, Mandalay Technological University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate 19087 EN Hyungjin Kim Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sho Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mizuki Kitamatsu Department of Applied Chemistry, Kindai University Kazunori Watanabe Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Takashi Ohtsuki Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G(1), S, and G(2)/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G(1)/S transition than in the G(1) and S/G(2)/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions. No potential conflict of interest relevant to this article was reported.

PUBLIC LIBRARY SCIENCE Acta Medica Okayama 1932-6203 14 1 2019 In-stem molecular beacon targeted to a 5 '-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity e0211505 EN Yuichi Miyoshi Graduate School of Natural Science and Technology, Okayama University Takashi Ohtsuki Graduate School of Natural Science and Technology, Okayama University Hiromu Kashida Graduate School of Engineering, Nagoya University Hiroyuki Asanuma Graduate School of Engineering, Nagoya University Kazunori Watanabe Graduate School of Natural Science and Technology, Okayama University Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2041-1723 7 2016 Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs 12501 EN Takashi Ohtsuki Department of Biomedical Engineering, Okayama University Shigeto Kanzaki Department of Biomedical Engineering, Okayama University Sae Nishimura Department of Biomedical Engineering, Okayama University Yoshio Kunihiro Department of Biomedical Engineering, Okayama University Masahiko Sisido Department of Biomedical Engineering, Okayama University Kazunori Watanabe Department of Biomedical Engineering, Okayama University The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2045-2322 5 2015 The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates. 18577 18577 EN Takashi Ohtsuki Department of Medical Bioengineering, Okayama University Shunya Miki Department of Medical Bioengineering, Okayama University Shouhei Kobayashi Advanced ICT Research Institute Kobe, NICT Tokuko Haraguchi Advanced ICT Research Institute Kobe, NICT Eiji Nakata Institute of Advanced Energy, Kyoto University Kazutaka Hirakawa Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University Kensuke Sumita Department of Medical Bioengineering, Okayama University Kazunori Watanabe Department of Medical Bioengineering, Okayama University Shigetoshi Okazaki Department of Medical Spectroscopy, Hamamatsu University School of Medicine In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated (1)O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of (1)O2 was confirmed using (1)O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than (1)O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2041-1723 7 2016 Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs 12501 12501 EN Takashi Ohtsuki Department of Biomedical Engineering, Okayama University Shigeto Kanzaki Department of Biomedical Engineering, Okayama University Sae Nishimura Department of Biomedical Engineering, Okayama University Yoshio Kunihiro Department of Biomedical Engineering, Okayama University Masahiko Sisido Department of Biomedical Engineering, Okayama University Kazunori Watanabe Department of Biomedical Engineering, Okayama University The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4 h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405 nm at 125 mW cm(-2), DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19 s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells. No potential conflict of interest relevant to this article was reported.