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Papers - Yamada Sota

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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

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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Development of a Fluorescence Probe for Detecting Nitroreductase Activity Based on Steric Repulsion-Induced Twisted Intramolecular Charge Transfer (sr-TICT)

Sugimoto M., Sasaki E., Ohno H., Ikeno T., Yamada S., Hanaoka K.

Chemical and Pharmaceutical Bulletin 72 ( 9 ) 810 - 816 2024.09

Research paper (scientific journal), Accepted, ISSN 00092363

Twisted intramolecular charge transfer (TICT) is a phenomenon involving intramolecular charge transfer together with intramolecular rotation upon photoexcitation, and in general this excited state of fluorescent dyes undergoes non-radiative decay (producing no fluorescence). We recently discovered that the magnitude of TICT in rhodamine derivatives could be regulated by altering the size of the substituents on the xanthene moiety, generating differing degrees of intramolecular steric repulsion. To further illustrate the usefulness and generality of this strategy, we describe here an application of quinone methide chemistry, which is widely used as a fluorescence off/on switching reaction for fluorescence probes detecting enzymatic activity, to construct a steric repulsion-induced (sr)-TICT-based fluorescence probe targeting nitroreductase (NTR) activity. The developed probe was almost non-fluorescent in phosphate-buffered saline (PBS) due to strong induction of the TICT state. On the other hand, when the probe was incubated with NTR and nicotinamide adenine dinucleotide (NADH), a large fluorescence increase was observed over time. We confirmed that the enzymatic reaction proceeded as expected, i.e., the nitro group of the probe was reduced to the corresponding amino group, followed by spontaneous elimination of iminoquinone methide. These results suggest that our simple design strategy based on the sr-TICT mechanism, i.e., controlling intramolecular steric repulsion, would be applicable to the development of fluorescence probes for a variety of enzymes.

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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

ISSN 14770520

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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Viral vector purification with thermoresponsive-anionic mixed polymer brush modified beads-packed column

Nagase K., Kitazawa S., Kogure T., Yamada S., Katayama K., Kanazawa H.

Separation and Purification Technology 286 120445 2022.04

Research paper (scientific journal), Accepted, ISSN 13835866

Viral vectors have attracted attention as a potential new therapeutic modality for gene therapy. In this study, we developed a temperature-modulated viral vector purification column using a mixed polymer brush composed of thermoresponsive and anionic polymers as packing material ligands. The mixed polymer brushes were modified on silica beads using a combination of reversible addition − fragmentation chain transfer (RAFT) polymerization of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) and subsequent atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). The temperature-modulated zeta potential change in the prepared mixed polymer brush was attributed to the PNIPAAm shrinking and exposing of PAMPS. The prepared mixed polymer brush modified beads were used as packing materials, and the elution behavior of the adeno associated virus type 2 (AAV2) vector was observed. The AAV2 vector was adsorbed on the mixed polymer brush by electrostatic and hydrophobic interactions at 40 °C. By reducing the temperature to 5 °C, adsorbed AAV2 vector on the mixed polymer brush was desorbed and eluted from the column due to lowering the electrostatic and hydrophobic interactions between the AAV2 vector and mixed polymer brush. The AAV2 vector was separated from bovine serum proteins as a contaminant using the column. The ability to infect cells was maintained by the recovered AAV2 vectors from the column. Thus, the developed column would be beneficial for the simple purification of viral vectors.

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Thermally-modulated cell separation columns using a thermoresponsive block copolymer brush as a packing material for the purification of mesenchymal stem cells

Nagase K., Edatsune G., Nagata Y., Matsuda J., Ichikawa D., Yamada S., Hattori Y., Kanazawa H.

Biomaterials Science 9 ( 21 ) 7054 - 7064 2021.11

Research paper (scientific journal), Accepted, ISSN 20474830

Cell therapy using mesenchymal stem cells (MSCs) is used as effective regenerative treatment. Cell therapy requires effective cell separation without cell modification and cellular activity reduction. In this study, we developed a temperature-modulated mesenchymal stem cell separation column. A temperature-responsive cationic block copolymer, poly(N,N-dimethylaminopropylacrylamide)-b-poly(N-isopropylacrylamide)(PDMAPAAm-b-PNIPAAm) brush with various cationic copolymer compositions, was grafted onto silica beads via two-step atom transfer radical polymerization. Using the packed beads, the elution behavior of the MSCs was observed. At 37 °C, the MSCs were adsorbed onto the column via both hydrophobic and electrostatic interactions with the PNIPAAm and PDMAPAAm segments of the copolymer brush, respectively. By reducing the temperature to 4 °C, the adsorbed MSCs were eluted from the column by reducing the hydrophobic and electrostatic interactions attributed to the hydration and extension of the PNIPAAm segment of the block copolymer brush. From the temperature-modulated adsorption and elution behavior of MSCs, a suitable DMAPAAm composition of the block copolymer brush was determined. Using the column, a mixture of MSC and BM-CD34+ cells was separated by simply changing the column temperature. The column was used to purify the MSCs, with purities of 78.2%, via a temperature change from 37 °C to 4 °C. Additionally, the cellular activity of the MSCs was retained throughout the column separation step. Overall, the obtained results show that the developed column is useful for MSC separation without cell modification and cellular activity reduction. This journal is

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Temperature-responsive spin column for sample preparation using an all-aqueous eluent

Nagase K., Ishizawa Y., Inoue M., Kokubun M., Yamada S., Kanazawa H.

Analytica Chimica Acta 1179 338806 2021.09

Research paper (scientific journal), Accepted, ISSN 00032670

We present a temperature-responsive spin column using an all-aqueous eluent. The method is intended as a simple sample preparation method for protein removal from serum, which is required for serum drug analysis. As packing materials for the spin column, we prepared two types of silica beads via surface-initiated radical polymerization. The large beads (diameter, 40–63 μm) were grafted with a temperature-responsive cationic copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide-co-n-butyl methacrylate) (P(NIPAAm-co-DMAPAAm-co-BMA)), and the small beads (diameter, 5 μm) were grafted with a temperature-responsive hydrophobic copolymer, P(NIPAAm-co-BMA). The beads were packed into the spin column as a double layer: P(NIPAAm-co-BMA) silica beads on the bottom and P(NIPAAm-co-DMAPAAm-co-BMA) silica beads on the top. The sample purification efficacy of the prepared spin column was evaluated on a model sample analyte (the antifungal drug voriconazole mixed with blood serum proteins). At 40 °C, the serum proteins and voriconazole were adsorbed on the prepared spin column via hydrophobic and electrostatic interactions. When the temperature was decreased to 4 °C, the adsorbed voriconazole was eluted from the column with the pure water eluent, while the serum proteins remained in the column. This temperature-responsive spin column realizes sample preparation simply by changing the temperature.

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Selective capture and non-invasive release of cells using a thermoresponsive polymer brush with affinity peptides

Nagase K., Shimura M., Shimane R., Hanaya K., Yamada S., Akimoto A.M., Sugai T., Kanazawa H.

Biomaterials Science 9 ( 3 ) 663 - 674 2021.02

Research paper (scientific journal), Accepted, ISSN 20474830

Tissue engineering and cell transplantation therapy have become promising therapies for intractable diseases. These approaches require cell separation technology without cell modification. Accordingly, in this study, we developed a novel cell separation method using a thermoresponsive block copolymer brush with an affinity peptide. A block copolymer brush with bottom poly(2-hydroxyethyl methacrylate [HEMA]-co-propargyl acrylate) and top poly(N-isopropylacrylamide-co-HEMA) segments was prepared through two steps of atom transfer radical polymerization. Then, cell affinity peptides were conjugated to the bottom segment of the copolymer brush through a click reaction. Using cRGD as a cell-Affinity peptide, enhancement of cell adhesion with rapid adhesion on the copolymer brush was observed at 37 °C, whereas the copolymer brush without cRGD did not exhibit cell adhesion. Temperature-modulated cell adhesion and detachment were performed with a relatively long upper segment because the affinity between peptides and cells was modulated by the swelling and shrinking of the upper thermoresponsive segment. Selective endothelial cell adhesion was performed at 37 °C using GGGREDV as an affinity peptide. Smooth muscle cells and fibroblasts did not adhere to the copolymer brush. Adhered human umbilical vein endothelial cells (HUVECs) were successfully recovered by reducing the temperature to 20 °C. Based on the properties of the copolymer brush, HUVECs could be purified using a mixture of cells simply by changing the temperature. These results demonstrated that the prepared copolymer brush with cell affinity peptides could be a useful cell separation tool because the cells could be separated with specificity and without cell modification using a simple procedure.

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Antibody drug separation using thermoresponsive anionic polymer brush modified beads with optimised electrostatic and hydrophobic interactions

Nagase K., Ishii S., Ikeda K., Yamada S., Ichikawa D., Akimoto A., Hattori Y., Kanazawa H.

Scientific Reports 10 ( 1 ) 11896 2020.12

Research paper (scientific journal), Accepted, ISSN 20452322

Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects. Thus, separation and analysis techniques for these antibody drugs have increased in importance. In the present study, we develop functional chromatography matrices for antibody drug separation and analysis. Three types of polymers, poly(N-isopropylacrylamide (NIPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-N-phenyl acrylamide (PhAAm)), P(NIPAAm-co-AMPS-co-n-butyl methacrylate (BMA)), and P(NIPAAm-co-AMPS-co-tert-butylacrylamide (tBAAm)), were modified on silica beads through atom transfer radical polymerisation. Rituximab elution profiles were observed using the prepared beads-packed column. Rituximab adsorption at high temperature and elution at low temperature from the column were observed, as a result of the temperature-modulated electrostatic and hydrophobic interactions. Using the column, rituximab purification from contaminants was performed simply by changing the temperature. Additionally, three types of antibody drugs were separated using the column through temperature-modulated hydrophobic and electrostatic interactions. These results demonstrate that the temperature-responsive column can be applied for the separation and analysis of biopharmaceuticals through a simple control of the column temperature.

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Thermoresponsive Cationic Block Copolymer Brushes for Temperature-Modulated Stem Cell Separation

Nagase K., Ota A., Hirotani T., Yamada S., Akimoto A., Kanazawa H.

Macromolecular Rapid Communications 41 ( 19 ) 2000308 2020.10

Research paper (scientific journal), Accepted, ISSN 10221336

Recently, cell separation methods have become important for preparing cells for transplantation therapy. In this study, a thermoresponsive cationic block copolymer brush is developed as an effective cell separation tool. This brush is prepared on glass surfaces using two steps of activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP). The cationic segment is prepared in the first step of the ARGET-ATRP of N,N-dimethylaminopropylacrylamide (DMAPAAm). In the second step, the thermoresponsive segment is prepared, attached to the bottom cationic segment, through ARGET-ATRP with N-isopropylacrylamide (NIPAAm). The cell adhesion behavior of the prepared thermoresponsive cationic copolymer, PDMAPAAm-b-PNIPAAm, brush is observed using umbilical cord-derived mesenchymal stem cells (UCMSC), fibroblasts, and macrophages. At 37 °C, all three types of cells adhere to the thermoresponsive cationic copolymer brush. Then, by reducing the temperature to 20 °C, the adhered UCMSC are detached from the copolymer brush, whereas the fibroblasts and macrophages remain adhered to the copolymer brush. Using this copolymer brush, UCMSC can be purified from the cell mixture simply by changing the temperature. Therefore, the prepared thermoresponsive cationic copolymer brush is useful as a cell separation tool for the purification of mesenchymal stem cells.

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Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions

Nagase K., Kitazawa S., Yamada S., Akimoto A., Kanazawa H.

Analytica Chimica Acta 1095 ( 6 ) 1 - 13 2020.01

Research paper (scientific journal), Accepted, ISSN 00032670

We developed a mixed polymer brush, which consists of temperature-responsive polymer and cationic polymer modified beads as functional chromatographic matrices, for temperature-modulated multiple hydrophobic and electrostatic interactions. The mixed polymer brush was modified on silica beads through the combination of reversible addition−fragmentation chain transfer (RAFT) polymerization of N,N-dimethylaminopropyl acrylamide (DMAPAAm) and surface initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). Zeta potential measurement of the mixed polymer brush revealed that the zeta potential increased with increasing temperature, which was attributed to the exposed PDMAPAAm on the beads arising from the shrinking of PNIPAAm upon increasing the temperature. The prepared beads were used as the packing material of high performance liquid chromatography (HPLC) columns, and the elution behavior of steroids, adenosine nucleotides, and proteins through the column was observed. The retention time of steroids increased with increasing the column temperature because of the dehydration of PNIPAAm in the mixed polymer brush. Adenosine nucleotides were also retained by the columns, which was attributed to the electrostatic interaction with PDMAPAAm in the mixed polymer brush. Several proteins were adsorbed on the column at elevated temperatures because of the enhanced electrostatic interaction of exposed PDMAPAAm and the enhanced hydrophobic interaction resulting from the dehydration of PNIPAAm. By exploiting this unique property, mixtures of proteins could be separated by simply changing the column temperature. These results indicate that the developed mixed polymer brush modified beads would be useful as functional chromatographic packing matrices for thermally-modulated multiple hydrophobic and electrostatic interactions.

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(7-Diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid as a caged inhibitor for photocontrol of histone deacetylase activity

Ieda N., Yamada S., Kawaguchi M., Miyata N., Nakagawa H.

Bioorganic & Medicinal Chemistry 24 ( 12 ) 2789 - 2793 2016.06

Research paper (scientific journal), Accepted, ISSN 09680896

Histone deacetylases (HDACs) are involved in epigenetic control of the expression of various genes by catalyzing deacetylation of ϵ-acetylated lysine residues. Here, we report the design, synthesis and evaluation of the (7-diethylaminocoumarin-4-yl)methyl ester of suberoylanilide hydroxamic acid (AC-SAHA) as a caged HDAC inhibitor, which releases the known pan-HDAC inhibitor SAHA upon cleavage of the photolabile (7-diethylaminocoumarin-4-yl)methyl protecting group in response to photoirradiation. A key advantage of AC-SAHA is that the caged derivative itself shows essentially no HDAC-inhibitory activity. Upon photoirradiation, AC-SAHA decomposes to SAHA and a 7-diethylaminocoumarin derivative, together with some minor products. We confirmed that AC-SAHA inhibits HDAC in response to photoirradiation in vitro by means of chemiluminescence assay. AC-SAHA also showed photoinduced inhibition of proliferation of human colon cancer cell line HCT116, as determined by MTT assay. Thus, AC-SAHA should be a useful tool for spatiotemporally controlled inhibition of HDAC activity, as well as a candidate chemotherapeutic reagent for human colon cancer.

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