Language：English   Publishing type：Research paper (scientific journal)  

In breast cancer treatment, the elimination of residual cancer cells in a sustainable and controllable manner to prevent recurrence remains a critical challenge in postoperative adjuvant therapy. In this study, a novel implantable in situ therapeutic composite scaffold platform (ALA@lipo/Au/Gel/PGA) was developed based on a porous scaffold composed of biocompatible gelatin and polyglutamic acid (PGA). This platform incorporated the photothermal agent Au nanorods (AuNRs) and thermosensitive liposomes encapsulated with the photosensitizer precursor 5-aminolevulinic acid (ALA). Due to the satisfactory photothermal conversion effect of the ALA@lipo/Au/Gel/PGA composite scaffold, the use of near-infrared (NIR) light not only ablated the cancer cells in the scaffold through photothermal therapy (PTT) but also induced the accelerated release of encapsulated ALA from the thermosensitive liposomes. After uptake, ALA could generate cytotoxic reactive oxygen species to increase tumour cell elimination efficiency via photodynamic therapy (PDT). Both in vitro and in vivo experiments demonstrated the synergistic anticancer effects of the composite scaffold. These results highlight the potential of this phototherapy-induced composite scaffold as a new synergistic treatment method for breast cancer.

DOI： 10.1039/d5mh00888c

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The colon possesses a unique physiological environment among human organs, where there is a highly viscous body fluid layer called the mucus layer above colonic epithelial cells. Dysfunction of the mucus layer not only contributes to the occurrence of colorectal cancer (CRC) but also plays an important role in the development of chemoresistance in CRC. Although viscosity is an essential property of the mucus layer, it remains elusive how viscosity affects chemoresistance in colon cancer cells. In this study, the influence of viscosity on their chemoresistance was elucidated by culturing colon cancer cells in media of different viscosities supplemented with doxorubicin (DOX). The viscosity range was adjusted from 99.4 mPa s to 776.6 mPa s by adding polyethylene glycol of different molecular weights in culture medium. Cell viability in the high viscosity medium was higher than that in the low viscosity medium. Expression of chemoresistance-related genes such as ABCC2 and ABCG2 increased when cells were cultured in the high viscosity medium. Furthermore, cell migration increased while proliferation decreased when cells were cultured in the high viscosity medium. The colon cancer cells cultured in the high viscosity medium exhibited high expression of p21 mRNA. The results suggested that viscosity could affect the resistance of colon cancer cells to DOX by regulating the expression of chemoresistance-related and proliferation-related genes.

DOI： 10.1039/d4tb02334j

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.actbio.2025.01.004

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bioactmat.2024.10.011

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/D5MH00317B

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.biomaterials.2024.122511

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Language：English   Publishing type：Research paper (scientific journal)  

Chemotherapy is one of the most common strategies for cancer treatment, whereas drug resistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism of emerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cells may contribute to drug resistance. Although it is well known that ECM plays an important role in orchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In this study, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogels with a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancer cells for a three-dimensional culture with different concentrations of DOX. The viability of the cells cultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cell viability decreased with DOX concentration when the cells were cultured in the same stiffness hydrogels. When DOX concentration was the same, breast cancer cells showed higher viability in high-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression of P-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels. The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOX by regulating the expression of chemoresistance-related genes.

DOI： 10.3390/gels10030202

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Publishing type：Research paper (scientific journal)   Publisher：Fortune Journals  

DOI： 10.26502/jsr.10020350

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Publishing type：Research paper (scientific journal)   Publisher：The Society for Free Radical Research Japan  

DOI： 10.3164/jcbn.24-88

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Publishing type：Research paper (scientific journal)   Publisher：OAE Publishing Inc.  

Magnetic hyperthermia uses magnetic nanoparticles (MNPs) for conversion of magnetic energy into thermal energy under an alternating magnetic field (AMF) to increase local temperature for ablation of cancer cells. The magnetic thermal capacity of MNPs not only depends on the intrinsic properties of MNPs but is also affected by the microenvironmental matrices surrounding the MNPs. In this study, the influence of agarose hydrogels and gelatin porous scaffolds on the magnetic thermal property and anticancer effect of Fe3O4 nanoparticles (NPs) were investigated with a comparison to free Fe3O4 NPs. Flower-like Fe3O4 NPs were synthesized and embedded in agarose hydrogels and gelatin porous scaffolds. Under AMF irradiation, the free Fe3O4 NPs had the best magnetic thermal properties and the most efficiently increased the local temperature to ablate breast cancer cells. However, the Fe3O4 NPs embedded in agarose hydrogels and gelatin porous scaffolds showed reduced magnetic-thermal conversion capacity, and the local temperature change was decreased in comparison to free Fe3O4 NPs during AMF irradiation. The gelatin porous scaffolds showed a higher inhibitory influence than the agarose hydrogels. The inhibitory effect of agarose hydrogels and gelatin porous scaffolds on magnetic-thermal conversion capacity resulted in a decreased anticancer ablation capacity to breast cancer cells during AMF irradiation. The Fe3O4 NP-embedded gelatin scaffolds showed the lowest anticancer effect. The results suggested that the matrices used to deliver MNPs could affect their performance, and appropriate matrices should be designed to maximize their therapeutic effect for biomedical applications.

DOI： 10.20517/microstructures.2023.46

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Photodynamic therapy (PDT) is a great potential anti-tumor therapy owing to its non-invasiveness and high spatiotemporal selectivity. However, systemically administered photosensitizers diffuse in the skin and the eyes for a long duration, which cause phototoxicity to bright light and sunlight. Therefore, following PDT, patients must avoid exposure of to light and sunlight to avoid this phototoxicity. In this study, we have developed a locally administered PDT using nano-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups (aHP) as a photosensitizer. The aHP, approximately 3.0 nm in diameter, adhered the negatively charged cell membrane via electrostatic interaction. The aHP localized to the endosome via cell adhesion and induced apoptosis upon 635 nm light irradiation. On being administered subcutaneously on the tumor, 30% of the injected aHP remained in the administered sites. However, low-molecular-weight hematoporphyrin dihydrochloride (HP) disappeared due to rapid diffusion. PDT with locally administered aHP showed a higher anti-tumor effect after light irradiation at 635 nm for three days compared to low-molecular-weight HP. Intraperitoneal administration of HP caused severe phototoxicity upon irradiation with ultraviolet A at 10 J cm-2, whereas aHP did not cause phototoxicity because its diffusion into the skin could be suppressed, probably due to the high-molecular weight of aHP. Therefore, locally administered PDT with aHP is a potential PDT having high therapeutic efficacy without phototoxicity.

DOI： 10.3390/pharmaceutics15082076

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.omtn.2023.07.038

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/d3tb01174g

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Recently, plasma membrane-targeted photodynamic therapy has attracted attention as an effective cancer immunotherapeutic strategy. However, the released photosensitizers do not only adhere to the plasma membrane but may also be internalized in the cytosol, in endosomes/lysosomes, hindering investigations of the effects of photosensitizers attached to the plasma membrane. In this study, we developed a cell culture dish with singlet oxygen-generating cell-adhesive glass surfaces that allows investigation of the effects of photosensitizers attached to the plasma membrane. For cell adhesion, poly[N-(3-aminopropyl)methacrylamide] conjugated with hematoporphyrin PA-HpD was immobilized on the glass surfaces. Singlet oxygen was produced from the PA-HpD-immobilized glass surface upon laser irradiation at 635 nm. When murine colon adenocarcinoma 26 (Colon-26) cells were cultured on the PA-HpD-immobilized surface, the cells were swollen and ruptured, leading to effective apoptotic cell death using laser irradiation at 635 nm. In addition, microvesicles of approximately 10 μm in diameter were released from the plasma membrane into the culture medium. These phenomena were due to the oxidation of lipids in the cellular membrane, caused by the plasma membrane-targeted photodynamic therapy. In contrast, these phenomena were not observed on poly[N-(3-aminopropyl)methacrylamide]-immobilized glass surfaces. These results indicate that cell culture dishes with singlet oxygen-generating cell-adhesive glass surfaces can be used to investigate fundamental mechanisms in plasma membrane-targeted photodynamic therapy.

DOI： 10.1016/j.freeradbiomed.2023.07.028

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Matrix stiffness has been disclosed as an essential regulator of cell fate. However, it is barely studied how the matrix stiffness affects stem cell functions when cell morphology changes. Thus, in this study, the effect of hydrogel stiffness on adipogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) with controlled morphology was investigated. Micropatterns of different size and elongation were prepared by a photolithographical micropatterning technique. The hMSCs were cultured on the micropatterns and showed a different spreading area and elongation following the geometry of the underlying micropatterns. The cells with controlled morphology were embedded in agarose hydrogels of different stiffnesses. The cells showed a different level of adipogenic differentiation that was dependent on both hydrogel stiffness and cell morphology. Adipogenic differentiation became strong when the cell spreading area decreased and hydrogel stiffness increased. Adipogenic differentiation did not change with cell elongation. Therefore, cell spreading area and hydrogel stiffness could synergistically affect adipogenic differentiation of hMSCs, while cell elongation did not affect adipogenic differentiation. A change of cell morphology and hydrogel stiffness was accompanied by actin filament alignment that was strongly related to adipogenic differentiation. The results indicated that cell morphology could affect cellular sensitivity to hydrogel stiffness. The results will provide useful information for the elucidation of the interaction of stem cells and their microenvironmental biomechanical cues.

DOI： 10.1021/acsabm.3c00159

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Various studies have suggested the presence of triacylglycerol in cyanobacteria, but no convincing evidence exists. We purified a substance co-migrating with triacylglycerol in thin-layer chromatography and determined its structure using mass spectrometry, gas chromatography, and 1H and 13C NMR. The major components were palmitoyl and stearoyl plastoquinols (acyl plastoquinol). Acyl plastoquinol has never been described before, although acyloxy derivative of plastoquione has been described as plastoquinone B. The level of acyl plastoquinol was 0.4% of the total lipids. We still do not have clear evidence for the presence of triacylglycerol. If present, the maximum triacylglycerol level must be at most 10% of acyl plastoquinol. The Synechocystis Slr2103 protein was suggested to synthesize triacylglycerol, but the product could be acyl plastoquinol. The possible roles of this novel compound in photosynthesis should be a new focus of research.

DOI： 10.1016/j.bbrc.2022.12.003

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Postsurgical treatment of breast cancer remains a challenge with regard to killing residual cancer cells and regenerating breast defects. To prepare composite scaffolds for postoperative use, gelatin is chemically modified with folic acid (FA) and used for hybridization with citrate-modified Fe3 O4 nanoparticles (Fe3 O4 -citrate NPs) to fabricate Fe3 O4 /gelatin composite scaffolds which pore structures are controlled by free ice microparticles. The composite scaffolds have large spherical pores that are interconnected to facilitate cell entry and exit. The FA-functionalized composite scaffolds have the ability to capture breast cancer cells. The Fe3 O4 /gelatin composite scaffolds possess a high capacity for magnetic-thermal conversion to ablate breast cancer cells during alternating magnetic field (AMF) irradiation. In addition, the composite scaffolds facilitate the growth and adipogenesis of mesenchymal stem cells. The composite scaffolds have multiple functions for eradication of residual cancer cells under AMF irradiation and for regeneration of resected adipose tissue when AMF is off.

DOI： 10.1002/adhm.202202604

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In recent years, the synergistic effect of photothermal therapy (PTT) and chemotherapy has been recognized as an effective strategy for cancer treatment. Controlling the PTT temperature and drug release profile is desirable for minimizing the unexpected damage to normal cells. In this study, a smart platform of stepwise PTT and chemotherapy has been developed by using composite porous scaffolds of biodegradable black phosphorus (BP) nanosheets, gold nanorods(AuNRs), doxorubicin (Dox)-encapsulated thermosensitive liposomes and biodegradable polymers. Under near-infrared (NIR) laser irradiation, the composite scaffolds could attain high and low local temperatures before and after BP degradation, respectively. Dox release from the composite scaffolds could be controlled by the temperature change. In vitro cell culture and in vivo animal experiments indicated that a strong synergistic effect of PTT and chemotherapy could be achieved at an early stage of treatment before BP degradation, and a mild hyperthermia effect was shown for chemotherapy in the late stage after BP degradation. Moreover, the composite scaffolds after the complete release of Dox could support the proliferation of mesenchymal stem cells. The composite scaffolds showed a synergistic effect of stepwise PTT and chemotherapy for breast cancer elimination and promoted stem cell activities after killing cancer cells.

DOI： 10.1039/d2bm01155g

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Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Colour Material  

DOI： 10.4011/shikizai.95.331

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Cell morphology has been widely investigated for its influence on the functions of normal cells. However, the influence of cell morphology on cancer cell resistance to anti-cancer drugs remains unclear. In this study, micropatterned surfaces were prepared and used to control the spreading area and elongation of human breast cancer cell line. The influences of cell adhesion area and elongation on resistance to doxorubicin were investigated. The percentage of apoptotic breast cancer cells decreased with cell spreading area, while did not change with cell elongation. Large breast cancer cells had higher resistance to doxorubicin, better assembled actin filaments, higher DNA synthesis activity and higher expression of P-glycoprotein than small breast cancer cells. The results suggested that the morphology of breast cancer cells could affect their resistance to doxorubicin. The influence was correlated with cytoskeletal organization, DNA synthesis activity and P-glycoprotein expression.

DOI： 10.3390/polym14142761

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Photothermal nanoparticles are important in photothermal therapy. Combining different nanoparticles can achieve a high photothermal capacity. In this study, composite nanoparticles composed of black phosphorus nanosheets (BPNSs) and gold nanostars (BP-AuNSs) were synthesized by using BPNSs as the reductant. AuNSs were deposited on the BPNSs. The BP-AuNSs were further hybridized with porous gelatin scaffolds to prepare gelatin-BP-AuNS composite scaffolds. The gelatin-BP-AuNS composite scaffolds promoted cell migration and distribution. The synergistic effects of the BPNSs and AuNSs endowed the gelatin-BP-AuNS composite scaffolds with excellent photothermal properties. The gelatin-BP-AuNS composite scaffolds eliminated cancer cells after near infrared laser exposure and supported the adipogenic differentiation of human mesenchymal stem cells. Thus, this gelatin-BP-AuNS composite scaffold holds promise for breast cancer therapy.

DOI： 10.1016/j.bioadv.2022.212938

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Biomimetic microenvironments are important for controlling stem cell functions. In this study, different microenvironmental conditions were investigated for the stepwise control of proliferation and chondrogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs). The hMSCs were first cultured in collagen porous sponges and then embedded with or without collagen hydrogels for continual culture under different culture conditions. The different influences of collagen sponges, collagen hydrogels, and induction factors were investigated. The collagen sponges were beneficial for cell proliferation. The collagen sponges also promoted chondrogenic differentiation during culture in chondrogenic medium, which was superior to the effect of collagen sponges embedded with hydrogels without loading of induction factors. However, collagen sponges embedded with collagen hydrogels and loaded with induction factors had the same level of promotive effect on chondrogenic differentiation as collagen sponges during in vitro culture in chondrogenic medium and showed the highest promotive effect during in vivo subcutaneous implantation. The combination of collagen sponges with collagen hydrogels and induction factors could provide a platform for cell proliferation at an early stage and subsequent chondrogenic differentiation at a late stage. The results provide useful information for the chondrogenic differentiation of stem cells and cartilage tissue engineering.

DOI： 10.3390/ijms23126406

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/admi.202101978

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Interconnected scaffolds are useful for promoting the chondrogenic differentiation of stem cells. Collagen scaffolds with interconnected pore structures were fabricated with poly(lactic acid-co-glycolic acid) (PLGA) sponge templates. The PLGA-templated collagen scaffolds were used to culture human bone marrow-derived mesenchymal stem cells (hMSCs) to investigate their promotive effect on the chondrogenic differentiation of hMSCs. The cells adhered to the scaffolds with a homogeneous distribution and proliferated with culture time. The expression of chondrogenesis-related genes was upregulated, and abundant cartilaginous matrices were detected. After subcutaneous implantation, the PLGA-templated collagen scaffolds further enhanced the production of cartilaginous matrices and the mechanical properties of the implants. The good interconnectivity of the PLGA-templated collagen scaffolds promoted chondrogenic differentiation. In particular, the collagen scaffolds prepared with large pore-bearing PLGA sponge templates showed the highest promotive effect.

DOI： 10.1088/1748-605X/ac61f9

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Localization of tumors during laparoscopic surgery is generally performed by locally injecting India ink into the submucosal layer of the gastrointestinal tract using endoscopy. However, the location of the tumor is obscured because of the black-stained surgical field and the blurring caused by India ink. To solve this problem, in this study, we developed a tissue-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups. To evaluate the ability of tissue-adhesive porphyrin in vivo, low-molecular-weight hematoporphyrin and tissue-adhesive porphyrin were injected into the anterior wall of the exposed stomach in rats. Local injection of low-molecular-weight hematoporphyrin into the anterior wall of the stomach was not visible even after 1 day because of its rapid diffusion. In contrast, the red fluorescence of the tissue-adhesive porphyrin was visible even after 7 days due to the electrostatic interactions between the positively-charged moieties of the polycation in the tissue-adhesive porphyrin and the negatively-charged molecules in the tissue. In addition, intraperitoneal injection of tissue-adhesive porphyrin in rats did not cause adverse effects such as weight loss, hepatic or renal dysfunction, or organ adhesion in the abdominal cavity. These results indicate that tissue-adhesive porphyrin is a promising fluorescent tissue-marking agent.

DOI： 10.3390/ijms23084218

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The use of nanoparticles has been investigated as a new cancer treatment. These can induce specific cytotoxicity in cancer cells. In particular, Au nanoparticles (AuNPs) have unique characteristics. The maximum absorption spectrum of AuNPs can be adjusted to modify their size or shape to absorb near-infrared light that can penetrate into tissue without photodamage. Thus, the combination of AuNPs and near-infrared light can be used to treat cancer in deep-seated organs. To obtain effective cancer-specific accumulation of AuNPs, we focused on porphyrin and synthesized a porphyrin-attached Au compound: Au-HpD. In this study, we investigated whether Au-HpD possesses cancer-specific accumulation and cytotoxicity. Intracellular Au-HpD accumulation was higher in cancer cells than in normal cells. In order to analyze the cytotoxicity induced by Au-HpD, cancer cells and normal cells were co-cultured in the presence of Au-HpD; then, they were subjected to 870 nm laser irradiation. We observed that, after laser irradiation, cancer cells showed significant morphological changes, such as chromatin condensation and nuclear fragmentation indicative of cell apoptosis. This strong effect was not observed when normal cells were irradiated. Moreover, cancer cells underwent cell apoptosis with combination therapy.

DOI： 10.3390/molecules27041238

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The treatment of melanoma requires not only the elimination of skin cancer cells but also skin regeneration to heal defects. To achieve this goal, a bifunctional composite scaffold of poly(DL-lactic-co-glycolic acid) (PLGA), collagen and black phosphorus nanosheets (BPNSs) was prepared by hybridizing a BPNS-embedded collagen sponge with a PLGA knitted mesh. The composite mesh increased the temperature under near-infrared laser irradiation. The incorporation of BPNSs provided the PLGA-collagen-BPNS composite mesh with excellent photothermal properties for the photothermal ablation of melanoma cells both in vitro and in vivo. The PLGA-collagen-BPNS composite mesh had high mechanical strength for easy handling. The PLGA-collagen-BPNS composite mesh facilitated the proliferation of fibroblasts, promoted the expression of angiogenesis-related genes and the genes of components of the extracellular matrix for skin tissue regeneration. The high mechanical strength, photothermal ablation capability and skin tissue regeneration effects demonstrate that the bifunctional PLGA-collagen-BPNS composite mesh is a versatile and effective platform for the treatment of melanoma and the regeneration of skin defects.

DOI： 10.1039/d1tb02366g

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Significance: Ischemia-reperfusion (IR) injury is a major component of severe damage in vascular occlusion during stroke, myocardial infarction, surgery, and organ transplantation, and is exacerbated by the excessive generation of reactive oxygen species (ROS), which occurs particularly during reperfusion. With the aging of the population, IR injury is becoming a serious problem in various organs, such as the kidney, brain, and heart, as well as in the mesenteric capillaries. Recent Advances: To prevent reperfusion injuries, natural and synthetic low-molecular-weight (LMW) antioxidants have been well studied. Critical Issues: However, these LMW antioxidants have various problems, including adverse effects due to excessive cellular uptake and their rapid clearance by the kidney, and cannot fully exert their potent antioxidant capacity in vivo. Future Directions: To overcome these problems, we designed and developed redox polymers with antioxidants covalently conjugated with them. These polymers self-assemble into nanoparticles in aqueous media, referred to as redox nanoparticles (RNPs). RNPs suppress their uptake into normal cells, accumulate at inflammation sites, and effectively scavenge ROS in damaged tissues. We had developed two types of RNPs: RNPN, which disintegrates in response to acidic pH; and RNPO, which does not collapse, regardless of the environmental pH. Utilizing the pH-sensitive and -insensitive characteristics of RNPN and RNPO, respectively, RNPs were found to exhibit remarkable therapeutic effects on various oxidative stress disorders, including IR injuries. Thus, RNPs are promising nanomedicines for use as next-generation antioxidants. This review summarizes the therapeutic impacts of RNPs in the treatment of kidney, cerebral, myocardial, and intestinal IR injuries. Antioxid. Redox Signal. 36, 70-80.

DOI： 10.1089/ars.2021.0103

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Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using in vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.

DOI： 10.1002/cbic.202100478

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Interconnected pore structures of scaffolds are important to control the cell functions for cartilage tissue engineering. In this study, collagen scaffolds with interconnected pore structures were prepared using poly(D,L-lactide-co-glycolide) (PLGA) sponges as sacrificial templates. Six types of PLGA sponges of different pore sizes and porosities were prepared by the solvent casting/particulate leaching method and used to regulate the interconnectivity of the collagen scaffolds. The integral and continuous templating structure of PLGA sponges generated well-interconnected pore structures in the collagen scaffolds. Bovine articular chondrocytes cultured in collagen scaffolds showed homogenous distribution, fast proliferation, high expression of cartilaginous genes and high secretion of cartilaginous extracellular matrix. In particular, the collagen scaffold templated by the PLGA sacrificial sponge that was prepared with a high weight ratio of PLGA and large salt particulates showed the most promotive effect on cartilage tissue formation. The interconnected pore structure facilitated cell distribution, cell-cell interaction and cartilage tissue regeneration.

DOI： 10.1039/d1tb01559a

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Breast cancer treatment needs to eradicate cancer cells and restore breast defects after surgical intervention. Herein, bifunctional composite scaffolds of black phosphorus nanosheets (BPNSs) and gelatin were designed to kill breast cancer cells and induce adipose tissue reconstruction. The composite scaffolds were prepared by hybridizing photothermal BPNSs with porous gelatin matrices by adding pre-prepared ice particles to precisely adjust their pore structures. The composite scaffolds had large, well-interconnected spherical pores, which allowed cell migration and infiltration. Hybridization with BPNSs increased the compression strength of the scaffolds. The composite scaffolds possessed a high photothermal conversion capacity that was dependent on the amount of BPNSs. The composite scaffold with a high amount of BPNSs could completely kill breast cancer cells in vitro and in vivo under laser irradiation. Moreover, cell culture and animal experiment results showed that the composite scaffolds promoted lipid oil droplet formation and upregulated the expression of adipogenesis-related genes when hMSCs were cultured in the scaffolds. The composite scaffolds could offer a facile platform to exert anticancer effects against breast cancer cells and promote the reconstruction of adipose tissue.

DOI： 10.1016/j.biomaterials.2021.120923

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Gene transfection has been widely studied due to its potential applications in tissue repair and gene therapy. Many studies have focused on designing gene carriers and developing novel transfection techniques. However, the influence of cell size, shape and elongation on gene transfection has rarely been investigated. In this study, poly(vinyl alcohol)-micropatterned surfaces were prepared to precisely manipulate the size, shape and elongation of mesenchymal stem cells, and the influences of these factors on gene transfection were investigated. Cell size showed a significant influence on gene transfection. Elongation could affect the gene transfection of large cells but not small cells. Cells with a large spreading area and high aspect ratio showed high transfection with exogenous plasmid DNA. In particular, the transfection efficiency was the highest in micropatterned cells with a spreading area of 5024 μm2 and an aspect ratio of 8 : 1. In contrast, cell shape had no significant influence on gene transfection. The different influences of cell size, shape and elongation were correlated with their respective impacts on cytoskeletal structures, cellular nanoparticle uptake and DNA synthesis. Cells with a large size and elongated morphology showed well-organized actin filaments with a high cellular modulus, therefore promoting cellular nanoparticle uptake and DNA synthesis. Cells with different shapes showed similarities in actin filament organization, cellular modulus, uptake capacity and DNA synthesis. The results suggest the importance of cell size and elongation in exogenous gene transfection and should provide useful information for gene transfection and gene therapy.

DOI： 10.1039/d1tb00815c

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BACKGROUND: Bivalent thrombin-binding aptamers (TBAs) have great potential for the treatment of thrombosis because they exhibit high anticoagulant activity, and their complementary single-stranded DNA (ssDNA) sequences work as an antidote. However, a design strategy for antidote sequences against bivalent aptamers has not been established. OBJECTIVES: To develop bivalent TBAs using M08, which exhibits higher anticoagulant activity than the previously reported exosite Ⅰ-binding DNA aptamers, such as HD1, an exosite Ⅱ-binding DNA aptamer (HD22) was linked to M08 with various types of linkers. In addition, short-length complementary ssDNAs were designed to neutralize the optimized bivalent aptamer effectively and rapidly. RESULTS: Among the bivalent aptamers of M08 linked to HD22 with various types of linkers, M08-T15-HD22 possessed approximately 5-fold higher anticoagulant activity than previously reported bivalent aptamers. To neutralize the activity of the 87-meric M08-T15-HD22, complementary ssDNA sequences with different lengths and hybridization segments were designed. The complementary sequence against the M08 moiety played a more important role in neutralizing than that against the HD22 moiety. Hybridization of the T15 linker in the M08-T15-HD22 with the A15 sequence in the antidote accelerated neutralization due to toehold-mediated strand displacement. Interestingly, some shorter-length antidotes showed higher neutralizing activity than the full complementary 87-meric antidote, and the shortest, 34-meric antidote, neutralized most effectively. CONCLUSIONS: A pair comprising an 87-meric bivalent TBA containing M08 and a 34-meric short-length antidote with high anticoagulant and rapid neutralizing activities was developed. This design strategy of the DNA sequence can be used for other bivalent DNA aptamers and their antidotes.

DOI： 10.1002/rth2.12503

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Cell chirality has been demonstrated to be important for controlling cell functions. However, it is not clear how the chirality of the extracellular microenvironment regulates cell adhesion and cytoskeletal structures and therefore affects gene transfection. In this study, the chirality of focal adhesions and the cytoskeleton of single human mesenchymal stem cells (hMSCs) was controlled by specially designed micropatterns, and its influence on gene transfection was investigated. Micropatterns with different cell adhesion areas and swirling stripe lines were prepared by micropatterning fibronectin on polystyrene surfaces. The chiral micropatterns induced the formation of chiral focal adhesions and chiral cytoskeletal structures. Gene transfection efficiency was enhanced with increasing adhesion area, while hMSCs on left-handed and right-handed swirling micropatterns showed the same level of gene transfection. When the swirling angle was changed from 0°, 30°, and 60° to 90°, the gene transfection efficiency at a swirling angle of 60° was the lowest. The influence of cell chirality on gene transfection was strongly associated with cellular uptake capacity, DNA synthesis and cytoskeletal mechanics. The results demonstrated that cytoskeletal swirling had a significant influence on gene transfection.

DOI： 10.1016/j.biomaterials.2021.120751

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Soft nanofibrous oligopeptide hydrogels consisting of self-assembled l- and d-form Fmoc-Phe-Phe-Cys networks photo-cross-linked by poly(ethylene glycol)-dimethacrylate, which are referred to as l- and d-gel, respectively, were developed for investigation of chirality effects on the undifferentiated state of mesenchymal stem cells. Encapsulated mesenchymal stem cells in d-gel showed slower growth and less spreading, resulting in higher maintenance of the undifferentiated state, compared to in l-gel. This indicates that d-form peptide materials might be useful as scaffold materials for regenerative medicine using stem cells.

DOI： 10.2116/analsci.20SCN05

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The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation.

DOI： 10.1080/14686996.2021.1978273

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Accumulating evidence indicates that stem cell fate can be regulated by mechanical properties of the extracellular matrix. Most studies have focused onthe influence of matrix elasticity and viscoelasticity on stem cell differentiation. However, how matrix viscosity affects stem cell differentiation has been overlooked. In this study, a biphasic gelatin solution/hydrogel system is used for 3D culture of human bone marrow-derived mesenchymal stem cells (MSCs) to investigate the influence of gelatin solution viscosity on simultaneous osteogenic and adipogenic differentiation at the same culture condition. Gelatin solution promotes cell proliferation, while its promotive effect decreases with the increase of viscosity. The influence of viscosity on osteogenic and adipogenic differentiation of MSCs shows opposite trends. A high-viscosity gelatin solution results in an increase of alkaline phosphatase (ALP) activity, calcium deposition, and expression of osteogenesis-related genes. On the other hand, in a low-viscosity gelatin solution, a lot of lipid vacuoles are formed and adipogenesis-related genes are highly expressed. The results indicate high viscosity is beneficial for osteogenic differentiation, while low viscosity is beneficial for adipogenic differentiation. These findings suggest the importance of matrix viscosity on stem cell differentiation in 3D microenvironments.

DOI： 10.1002/adhm.202000617

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The chromatophores found in the cells of photosynthetic Paulinella species, once believed to be endosymbiotic cyanobacteria, are photosynthetic organelles that are distinct from chloroplasts. The chromatophore genome is similar to the genomes of α-cyanobacteria and encodes about 1,000 genes. Therefore, the chromatophore is an intriguing model of organelle formation. In this study, we analyzed the lipids of Paulinella micropora MYN1 to verify that this organism is a composite of cyanobacterial descendants and a heterotrophic protist. We detected glycolipids and phospholipids, as well as a betaine lipid diacylglyceryl-3-O-carboxyhydroxymethylcholine, previously detected in many marine algae. Cholesterol was the only sterol component detected, suggesting that the host cell is similar to animal cells. The glycolipids, presumably present in the chromatophores, contained mainly C16 fatty acids, whereas other classes of lipids, presumably present in the other compartments, were abundant in C20 and C22 polyunsaturated fatty acids. This suggests that chromatophores are metabolically distinct from the rest of the cell. Metabolic studies using isotopically labeled substrates showed that different fatty acids are synthesized in the chromatophore and the cytosol, which is consistent with the presence of both type I and type II fatty acid synthases, supposedly present in the cytosol and the chromatophore, respectively. Nevertheless, rapid labeling of the fatty acids in triacylglycerol and phosphatidylcholine by photosynthetically fixed carbon suggested that the chromatophores efficiently provide metabolites to the host. The metabolic and ultrastructural evidence suggests that chromatophores are tightly integrated into the whole cellular metabolism.

DOI： 10.1093/pcp/pcaa011

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Vascular endothelial growth factors (VEGFs) are hypoxia-inducible secreted proteins to promote angiogenesis, in which VEGF-A is an important molecule that binds and activates VEGF receptor-1 (VEGFR-1) and VEGFR-2. In this study, two DNA aptamers, Apt01 and Apt02, were successfully isolated by alternating consecutive systematic evolution of ligands by exponential enrichment (SELEX) against VEGFR-1 and -2 using deep sequencing analysis in an early selection round. Their binding affinities for VEGFR-2 were lower than that of VEGFR-1, which is similar to that of VEGF-A. Structural analyses with the measurements of circular dichroism spectra and ultraviolet melting curve showed that Apt01 possessed the stem-loop structure in the molecule, whereas Apt02 formed G-quadruplex structures. In addition, Apt02 accelerated a tube formation of human umbilical vein endothelial cells faster than Apt01, which was affected by difference of binding affinity and nuclease resistance due to G-quadruplex structures. These results demonstrated that Apt02 might have a potential to function as an alternative to VEGF-A.

DOI： 10.1016/j.omtn.2019.12.034

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Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C. debaryana was encapsulated in a Ca2+-alginate gel for the culture with high cell density. We discovered that the growth of the encapsulated cells resulted in the formation of spherical palmelloid colonies with high cell density, where daughter cells with truncated flagella remained wrapped within the mother cell walls. Interestingly, gel encapsulation markedly promoted proliferation of C. debaryana cells, and the encapsulated cells reached the stationary phase earlier than that of the free-living cells. Gel encapsulation also enhanced TAG accumulation. Gene expression analysis revealed that two genes of acyltransferases, DGAT1 and DGTT3, were upregulated in the stationary phase of free-living C. debaryana. In addition, the gene expression of these acyltransferases increased earlier in the encapsulated cells than that in the free-living cells. The enhanced production of TAG by alginate gel encapsulation was not found in C. reinhardtii which is known to use a different repertoire of acyltransferases in lipid accumulation.

DOI： 10.1093/pcp/pcz188

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The unicellular green microalga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin under stress conditions such as nitrogen deficiency. In this study, we discovered an astaxanthin accumulation in H. pluvialis cells by the addition of a synthetic cationic polymer, polyethyleneimine (PEI), into the cell culture. With an increase in PEI amount, amount of astaxanthin accumulation was increased. To investigate the mechanism for the accumulation of astaxanthin by the addition of PEI in H. pluvialis cells, we measured a localization of PEI in the cells and a production of reactive oxygen species. PEI was internalized in the cells through the negatively-charged cell walls, leading to excessive production of reactive oxygen species in the cells. Thus, the increased oxidative stress by cellular uptake of PEI resulted in the acceleration of astaxanthin accumulation in H. pluvialis.

DOI： 10.1016/j.jbiosc.2019.06.002

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We present a rapidly neutralizable and highly anticoagulant thrombin-binding aptamer with a short toehold sequence, originally discovered by systematic evolution of ligands by exponential enrichment (SELEX) with microbead-assisted capillary electrophoresis (MACE). MACE is a novel CE-partitioning method for SELEX and able to separate aptamers from a library of unbound nucleic acids, where the aptamer and target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. Three selection rounds of MACE-SELEX discovered several TBAs with a nanomolar affinity (Kd = 4.5-8.2 nM) that surpasses previously reported TBAs such as HD1, HD22, and NU172 (Kd = 118, 13, and 12 nM, respectively). One of the obtained aptamers, M08, showed a 10- to 20-fold longer prolonged clotting time than other anticoagulant TBAs, such as HD1, NU172, RE31, and RA36. Analyses of the aptamer and thrombin complexes using both bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX because of increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer and antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.

DOI： 10.1016/j.omtn.2019.03.002

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Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library. After only three selection rounds, we successfully enriched the modified aptamers and they showed slower off-rate than reported aptamers, suggesting MACE-SELEX is a promising approach for rapid identification of modified aptamers.

DOI： 10.2116/analsci.18SDN04

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Here, we demonstrated a strategy for developing signaling aptamers, based on screening of signaling aptamers from multiple aptamer candidates obtained by SELEX with next generation sequencing. Among aptamer candidates labelled by 6-carboxyfluorescein and quencher at both end termini, there is the possibility of discovering a potent signaling aptamer. In this study, we discovered DNA signaling aptamers against VEGFR-1. This strategy has the potential for signaling aptamer discovery without the extremely laborious task of optimization of oligodeoxynucleotide modifications.

DOI： 10.2116/analsci.18SDN05

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Chirality effects on stem cell fate were investigated in three-dimensional culture using soft fibrous hydrogels consisting of self-assembled l- and d-form Fmoc-Phe-Phe-Cys networks photo-cross-linked by poly(ethylene glycol) (l- and d-gel, respectively). Encapsulated human bone marrow-derived mesenchymal stem cells were all alive, spread, and grown in both hydrogels. Interestingly, the cells preferably spread and grew in l-gel compared to in d-gel under mixed induction, and cell osteogenesis and adipogenesis differentiation in d-gel were suppressed compared to in l-gel. These results revealed that stem cell function and fate can be regulated in three-dimensional hydrogel culture systems with chiral motifs.

DOI： 10.1021/acsabm.8b00123

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Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes. Upon application of an electric field, HL-60 cells as models of promyelocytes aggregated and floated between the saw-shaped electrodes, while UE7T-13 cells as models of BMSCs were effectively captured on the tips of the saw-shaped electrodes. After washing out the HL-60 cells from the device selectively, the purity of the UE7T-13 cells was increased from 33% to 83.5% within 5 min. Although further experiments and optimization are required, these results show the potential of the DEP device as a label-free rapid cell isolation system yielding high purity for rare and precious cells such as BMSCs.

DOI： 10.3390/s18093007

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Since much attention has been paid to in vivo biological functions of G-quadruplexes, structural analyses of G-quadruplexes are essential for understanding their functional mechanisms. Here, we established a simple optical-spectroscopy-based method for the estimation of G-quartet-forming guanines in parallel-type G-quadruplexes using measurements of circular dichroism and the thermal melting temperature.

DOI： 10.1039/c8an01131a

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A fingerprint-based sensing approach was used to characterize in vitro cellular senescence. Secretion profiles of cultured human fibroblasts in different senescent stages were transformed into colorimetric enzyme-activity fingerprints by applying cell culture media to a polyion complex array. Analysis of the obtained fingerprints using pattern recognition methods, such as linear discriminant analysis and hierarchical clustering analysis, revealed that the polyion complex array allows the noninvasive tracking of the replicative senescence progress even in those stages where a conventional marker such as senescence-associated β-galactosidase is negative. This fingerprint-based approach should thus offer an effective way for the routine monitoring or screening of in vitro cell senescence studies.

DOI： 10.1021/acs.analchem.8b00795

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Poly(ethylene sodium phosphate) (PEP·Na) showed excellent cytocompatibility and in vivo bone affinity. Moreover, PEP·Na did not interact with thrombin, which is a coagulation-related protein. Because immobilization of therapeutic agents and imaging probes on PEP·Na is easily performed, PEP·Na is a promising polymer for bone-targeted therapies.

DOI： 10.1039/c7bm00930e

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PURPOSE: Although nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) scavenge free radicals, their short half-life and considerable side effects such as systemic hypotension and bradycardia have limited their clinical application. Since a radical-containing nanoparticle (RNP) delivers nitroxyl radicals with a prolonged half-life specific to ischemic hearts, we investigated whether RNPs reduce infarct size without the occurrence of substantial side effects and whether nitric oxide (NO) contributes to the cardioprotective effects of RNPs. METHODS: The left anterior descending coronary arteries of dogs were occluded for 90 min, followed by reperfusion for 6 h. Either RNPs, micelles (not containing TEMPO) (control), or 4-hydroxy-TEMPO (TEMPOL) was injected into a systemic vein for 5 min before reperfusion. We evaluated the infarct size, myocardial apoptosis, plasma NO levels in coronary venous blood, and the RNP spectra using an electron paramagnetic resonance assay. RESULTS: RNPs reduced infarct size compared with the control group and TEMPOL group (19.5 ± 3.3 vs. 42.2 ± 3.7 vs. 30.2 ± 3.4%). RNPs also reduced myocardial apoptosis compared with the control and TEMPOL group. Coronary venous NO levels increased in the RNP group. CONCLUSIONS: In conclusion, the administration of 2,2,6,6-tetramethylpiperidine-1-oxyl as a RNP exerted cardioprotective effects against ischemia and reperfusion injury in canine hearts without exerting unfavorable hemodynamic effects. RNPs may represent a promising new therapy for patients with acute myocardial infarction.

DOI： 10.1007/s10557-017-6758-6

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Lineage commitment of stem cells is mainly regulated by their microenvironments, which comprise soluble growth factors, extracellular matrix, mechanical forces, and cell density. Although numerous studies have investigated stem cell response to these factors in two-dimensional (2D) culture, little is known about that in 3D culture. Here, we studied effects of 3D cell accumulation levels on the differentiation behavior of mesenchymal stem cells (MSCs) by using a micropatterned surface. After induction of 3D-cultured MSCs on the surface, their osteogenic differentiation was significantly promoted, while adipogenic differentiation was not. This differentiation behavior of densely packed MSCs in 3D culture is unlike that in 2D culture. Moreover, to determine the contributing factor of this commitment, the relationship between 3D cell accumulation levels and their differentiation potential was studied before differentiation induction. A series of MSCs with varied 3D accumulation levels were constructed on the micropatterned surface, where the accumulated MSCs were not in hypoxic environment. Interestingly, with increasing 3D accumulation levels, MSCs enhanced their osteogenic potential but repressed adipogenic potential in the gene expression level. These results suggest that preconditioned 3D microenvironments with high cell accumulation levels promote osteogenic differentiation of MSCs and their accumulation levels help in regulating MSC differentiation.

DOI： 10.1021/acsami.6b15688

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UV rays induce melanin production in the skin, which, from a cosmetic point of view, is problematic. Reactive oxygen species (ROS) generated in the skin upon UV irradiation are thought to be responsible for melanin production. Thus, effective antioxidants are recognized as useful tools for prevention of UV-induced melanin production. Redox nanoparticles (RNPs) containing nitroxide radicals as free radical scavengers were previously developed, and shown to be effective ROS scavengers in the body. RNPs are therefore expected to be useful for effective protection against UV-induced melanin production. However, as the sizes of RNPs are typically larger than the intercellular spaces of the skin, transdermal penetration is difficult. We recently demonstrated effective transdermal delivery and accumulation of nanoparticles in the epidermal layer via faint electric treatment, i.e., iontophoresis, suggesting that iontophoresis of RNPs may be a useful strategy for prevention of UV-induced melanin production in the skin. Herein, we performed iontophoresis of RNPs on the dorsal skin of hairless mice that produce melanin in response to light exposure. RNPs accumulated in the epidermal layer upon application of iontophoresis. Further, the combination of RNPs with iontophoresis decreased UV-induced melanin spots and melanin content in the skin. Taken together, we successfully demonstrated that iontophoresis-mediated accumulation of RNPs in the epidermis prevented melanin production.

DOI： 10.1248/bpb.b17-00155

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Pioglitazone is a widely used anti-diabetic drug that induces cytotoxicity in cancer cells; however, its clinical use is questioned due to its associated liver toxicity caused by increased oxidative stress. We therefore employed nitroxide-radical containing nanoparticle, termed redox nanoparticle (RNP(N)) which is an effective scavenger of reactive oxygen species (ROS) as a drug carrier. RNP(N) encapsulation increased pioglitazone solubility, thus increasing cellular uptake of encapsulated pioglitazone which reduced the dose required to induce toxicity in prostate cancer cell lines. Investigation of in vitro molecular mechanism of pioglitazone revealed that both apoptosis and cell cycle arrest were involved in tumor cell death. In addition, intravenously administered pioglitazone-loaded RNP(N) produced significant tumor volume reduction in vivo due to enhanced permeation and retention effect. Most importantly, oxidative damage caused by pioglitazone in the liver was significantly suppressed by pioglitazone-loaded RNP(N) due to the presence of nitroxide radicals. It is interesting to note that oral administration of encapsulated pioglitazone, and co-administration of RNP(N) and pioglitazone, i.e., no encapsulation of pioglitazone in RNP(N) also significantly contributed to suppression of the liver injury. Therefore, use of RNP(N) either as an adjuvant or as a carrier for drugs with severe side effects is a promising chemotherapeutic strategy.

DOI： 10.1016/j.biomaterials.2016.05.001

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In this study, we investigated the effect of positive dielectrophoresis (DEP) on gene expression in mesenchymal stem cells. When applying an alternating current voltage, human bone marrow derived mesenchymal stem cells (UE7T-13) exhibited a positive DEP, and were compressed onto the electrode surface. The constructed device can easily control the DEP force to the cells by changing the frequency. Interestingly, gene expressions of the cell differentiation marker in UE7T-13 cells and the mechanical stimulation-susceptible one were changed by applying a positive DEP. These results suggested that the gene expression in mesenchymal stem cells can be regulated by applying mechanical stimulation derived from DEP.

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Curcumin is a phytochemical with diverse molecular targets and is well known for its anti-tumor potential. However, it has limited application in cancer therapy because curcumin undergoes rapid oxidative degradation at physiological conditions resulting in poor stability and bio-availability. In this study, we were able to suppress curcumin's oxidative degradation by encapsulating it in a nanoparticle that also acts as a radical scavenger. We prepared curcumin-loaded pH-sensitive redox nanoparticles (RNP(N)) by self-assembling amphiphilic block copolymers conjugated with reactive oxygen species (ROS) scavenging nitroxide radicals to ensure the delivery of minimally degraded curcumin to target regions. In vitro analysis confirmed that the entrapment of both curcumin and nitroxide radicals in the hydrophobic core of RNP(N) suppressed curcumin degradation in conditions mimicking the physiological environment. Evaluation of apoptosis-related molecules in the cells, such as ceramides, caspases, apoptosis-inducing factor, and acid ceramidase revealed that curcumin loaded RNP(N) induced strong apoptosis compared to free curcumin. Lastly, intravenous injection of curcumin loaded RNP(N) suppressed tumor growth in vivo, which is due to the increased bio-availability and significant ROS scavenging at tumor sites. These results demonstrated that RNP(N) is a promising drug carrier with unique ROS-scavenging abilities, and it is able to overcome the crucial hurdle of curcumin's limitations to enhance its therapeutic potential.

DOI： 10.1016/j.jconrel.2015.04.025

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Oral chemotherapy is the preferred treatment for colon cancer. However, this strategy faces many challenges, including instability in the gastrointestinal (GI) tract, insufficient bioavailability, low tumor targeting, and severe adverse effects. In this study, we designed a novel redox nanoparticle (RNP(O)) that is an ideal oral therapeutics for colitis-associated colon cancer treatment. RNP(O) possesses nitroxide radicals in the core, which act as reactive oxygen species (ROS) scavengers. Orally administered RNP(O) highly accumulated in colonic mucosa, and specifically internalized in cancer tissues, but less in normal tissues. Despite of long-term oral administration of RNP(O), no noticeable toxicities were observed in major organs of mice. Because RNP(O) effectively scavenged ROS, it significantly suppressed tumor growth after accumulation at tumor sites. Combination of RNP(O) with the conventional chemotherapy, irinotecan, led to remarkably improved therapeutic efficacy and effectively suppressed its adverse effects on GI tract. Therefore, RNP(O) is promising oral nanotherapeutics for cancer therapies.

DOI： 10.1016/j.biomaterials.2015.03.037

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Silica-containing redox nanoparticles (siRNP) are nanocomposites consisting of silica nanoparticles and amphiphilic block copolymers with nitroxide radicals as reactive oxygen species (ROS) scavengers. Electrostatic interactions between the cationic segment of a polymer in the core and the entrapped silica nanoparticles form a crosslinking structure that provides siRNP stability in vivo, even under harsh conditions in the gastrointestinal tract. Due to the adsorption character of silica nanoparticles in the nanocomposite, siRNP can be applied not only for adsorbents of body wastes but also for drug carriers with high loading capacity. The ROS-scavenging character of siRNP significantly improves their performance for medical applications. Here, we describe the development of siRNP and provide two examples of their medical applications as (1) novel nano-sized adsorbents for peritoneal dialysis, and (2) orally administrable drug carriers for the treatment of gastrointestinal inflammation.

DOI： 10.1039/c5bm00057b

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Excessively generated reactive oxygen species are associated with age-related neurodegenerative diseases. We investigated whether scavenging of reactive oxygen species in the brain by orally administered redox nanoparticles, prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals, facilitates the recovery of cognition in 17-week-old senescence-accelerated prone (SAMP8) mice. The redox polymer was delivered to the brain after oral administration of redox nanoparticles via a disintegration of the nanoparticles in the stomach and absorption of the redox polymer at small intestine to the blood. After treatment for one month, levels of oxidative stress in the brain of SAMP8 mice were remarkably reduced by treatment with redox nanoparticles, compared to that observed with low-molecular-weight nitroxide radicals, resulting in the amelioration of cognitive impairment with increased numbers of surviving neurons. Additionally, treatment by redox nanoparticles did not show any detectable toxicity. These findings indicate the potential of redox polymer nanotherapeutics for treatment of the neurodegenerative diseases.

DOI： 10.1371/journal.pone.0126013

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AIM: Oxidative stress (OS) is largely thought to be a central mechanism responsible for liver damage, inflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Our aim was to investigate whether suppression of OS in the liver via redox nanoparticles (RNPs) reduces liver damage in a mouse model of NASH. MATERIALS & METHODS: RNPs were prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals and were orally administered by daily gavage for 4 weeks. RESULTS: The redox polymer was delivered to the liver after disintegration of nanoparticle in the stomach. RNP treatment in NASH mice via gavage led to a reduction of liver OS, improvement of fibrosis, and significant reduction of inflammation. CONCLUSION: These findings uncover RNP as a novel potential NASH therapy.

DOI： 10.2217/nnm.15.87

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This Progress Report describes a development of two types of reactive oxygen species (ROS)-scavenging nanomedicines for the treatment of oxidative stress injuries, referred to as pH-sensitive redox nanoparticle (RNP(N) ) and pH-insensitive redox nanoparticle (RNP(O) ), which are prepared by self-assembling amphiphilic block copolymers possessing nitroxide radicals as a side chain of hydrophobic segment via amine and ether linkages, respectively. Due to a protonation of amino groups in hydrophobic core, RNP(N) disintegrates in low pH environments such as ischemic, inflamed, and tumor tissues, resulting in increased ROS-scavenging activity because of the exposed nitroxide radicals from the core. Utilizing pH-responsiveness of RNP(N) , it shows remarkable therapeutic effects on oxidative stress injuries such as renal and cerebral ischemia-reperfusion injuries after intravenous administration. Moreover, RNP(N) shows an enhancement of the activity of anticancer drugs by suppression of activation of transcription factors in tumor due to the ROS scavenging. On the other hand, orally administered RNP(O) has notable characteristics such as preferential accumulation in mucosa and inflamed area of gastrointestinal tract and no uptake into blood stream. Based on these characters, RNP(O) shows a remarkable therapeutic effect for the gastrointestinal inflammation without any adverse effects. Thus, ROS-scavenging nanomedicines have therapeutic efficacy in numerous oxidative stress diseases.

DOI： 10.1002/adhm.201300576

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We developed a nitroxide radicals-containing polymer (NRP), which is composed of poly(4-methylstyrene) possessing nitroxide radicals as a side chain via amine linkage, to scavenge reactive oxygen species (ROS) from cigarette smoke. In this study, the NRP was coated onto cigarette filters and its ROS-scavenging activity from streaming cigarette smoke was evaluated. The intensity of electron spin resonance signals of the NRP in the filter decreased after exposure to cigarette smoke, indicating consumption of nitroxide radicals. To evaluate the ROS-scavenging activity of the NRP-coated filter, the amount of peroxy radicals in an extract of cigarette smoke was measured using UV-visible spectrophotometry and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The absorbance of DPPH at 517 nm decreased with exposure to cigarette smoke. When NRP-coated filters were used, the decrease in the absorbance of DPPH was prevented. In contrast, both poly[4-(cyclohexylamino)methylstyrene]- and poly(acrylic acid)-coated filters, which have no nitroxide radical, did not show any effect, indicating that the nitroxide radicals in the NRP scavenge the ROS in cigarette smoke. As a result, the extract of cigarette smoke passed through the NRP-coated filter has a lower cellular toxicity than smoke passed through poly[4-(cyclohexylamino)methylstyrene]- and poly(acrylic acid)-coated filters. Accordingly, NRP is a promising material for ROS scavenging from cigarette smoke.

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Antioxidants have been demonstrated to exert beneficial effects as pharmacotherapies for cardiovascular diseases. The in vitro systems generally employed to evaluate antioxidants, however, are limited by having no appreciable in vivo redox status of the antioxidants. Therefore, we used our developing chicken egg model to evaluate the in vivo antioxidative activity of a redox nanoparticle possessing 2,2,6,6-tetramethylpiperidine-1-oxyl (RNP(O)). The 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) elicited strong oxidative stress and its LD50 value for chick embryos was 3.5±0.9mg/egg. The low molecular weight nitroxide compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), which is known to have the highest level of antioxidant activity, showed no significant protective effect against AAPH-induced embryo lethality. On the contrary, RNP(O) had potent protective effects against AAPH-induced embryo lethality. Moreover, RNP(O) could significantly suppress the production of lipid peroxides in chick serum induced by hydrocortisone. Since RNP(O) has a longer retention time in blood than TEMPOL, RNP(O) may protect the embryo against lethal oxidative stress by suppressing lipid peroxidation. The validity of in vivo experiments using developing chicken eggs was supported by our data, where RNP(O) was determined to elicit strong antioxidative activity in vivo, irrespective of the lack of a significant difference in the in vitro activity between low-molecular weight TEMPOL and RNP(O). Our results support the use of the developing chicken egg model to evaluate the potential in vivo antioxidative activity of RNP(O).

DOI： 10.1016/j.jconrel.2014.03.015

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BACKGROUND: Patients with ulcerative colitis (UC) exhibit overproduction of reactive oxygen species (ROS) and imbalance of colonic microflora. We previously developed a novel redox nanoparticle (RNP(O)), which effectively scavenged ROS in the inflamed mucosa of mice with dextran sodium sulfate (DSS)-induced colitis after oral administration. The objective of this study was to examine whether the orally administered RNP(O) changed the colonic microflora in healthy mice and those with colitis. METHODS: RNP(O) was synthesized by self-assembly of an amphiphilic block copolymer that contains stable nitroxide radicals in hydrophobic side chain via ether linkage. Colitis was induced in mice by supplementing DSS in drinking water for 7 days, and RNP(O) was orally administered daily during DSS treatment. The alterations of fecal microflora during treatment of DSS and RNP(O) were investigated using microbiological assays. RESULTS: We investigated that RNP(O) did not result in significant changes to the fecal microflora in healthy mice. Although total aerobic and anaerobic bacteria were not significantly different between experimental groups, a remarkable increase in commensal bacteria (Escherichia coli and Staphylococcus sp.) was observed in mice with DSS-induced colitis. Interestingly, orally administered RNP(O) remarkably reduced the rate of increase of these commensal bacteria in mice with colitis. CONCLUSIONS: On the basis of the obtained results, it was confirmed that the oral administration of RNP(O) did not change any composition of bacteria in feces, which strongly suggests a protective effect of RNP(O) on healthy environments in intestinal microflora. RNP(O) may become an effective and safe medication for treatment of UC.

DOI： 10.1007/s00535-013-0836-8

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Here, we report an interaction between blood and redox nanoparticles, prepared by self-assembly of amphiphilic block copolymers possessing 2,2,6,6-tetramethylpiperidine-N-oxyls as a side chain of hydrophobic segment. When 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl was added to rat whole blood, its electron spin resonance signal disappeared rapidly. In contrast, the signal from redox nanoparticles remained for a long period of time, indicating that nitroxide radicals were protected in the blood by their compartmentalization in the core of nanoparticle. Although most 2,2,6,6-tetramethylpiperidine-N-oxyls were located in the nanoparticle core, reactive oxygen species-scavenging activity was found outside of blood cells. For example, redox nanoparticles suppressed superoxide anion-induced hemolysis effectively, while 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl did not. It was revealed that redox nanoparticles were not internalized into the healthy blood cells, which was in sharp contrast to 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. Due to its internalization into healthy platelets, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl induced mitochondrial dysfunction, while redox nanoparticles did not. Redox nanoparticles suppressed platelet adhesion and extended blood coagulation time, in contrast to 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. These results indicate that redox nanoparticles scavenge reactive oxygen species outside of cells, but do not interfere with normal redox reactions inside of the cell. Based on these results, we determine that an anti-oxidative strategy based on nanotechnology is a rational and safe therapeutic approach.

DOI： 10.3164/jcbn.13-85

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The prevention of encapsulating peritoneal sclerosis (EPS) and the enhancement of dialysis efficiency are two important strategies that can improve the quality of life of patients undergoing peritoneal dialysis. We have thus far developed bionanoparticles that effectively scavenge reactive oxygen species (redox nanoparticles; RNPs). The objective of this study was to apply RNPs as a component of dialysate to reduce oxidative stress. Porous silica nanoparticles were combined with RNPs to enhance the effective adsorption capacity of low-molecular weight (LMW) compounds. The silica-containing RNPs (siRNPs) were confirmed to statistically decrease the level of creatinine and blood urea nitrogen in vivo. EPS model rats that underwent an intraperitoneal injection of chlorhexidine gluconate exhibited dysfunction of the peritoneal membrane. siRNP administration did not result in dysfunction of the peritoneal membrane. An LMW nitroxide compound, TEMPOL, also showed a weak peritoneal protective effect, although its efficiency was limited. No blood uptake of siRNPs was observed when they were administered into the peritoneal cavity. However, LMW-TEMPOL diffused into the blood stream, which might have decreased its effective concentration in the peritoneal cavity and led to adverse effects across the entire body. Considering these results, siRNPs are expected to be a new multi-functional nanomaterial for high performance peritoneal dialysis.

DOI： 10.1039/c3bm60236b

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BACKGROUND: Continuous administration of low-dose nonsteroidal anti-inflammatory drugs such as indomethacin (IND) is associated with an increased risk of gastrointestinal damage. In this study, the authors developed IND-loaded redox nanoparticles (IND@RNP(O)) with core-shell-type polymeric micelles possessing nitroxide radicals as reactive oxygen species scavengers. RESULTS: Orally administered IND@RNP(O) significantly accumulated in the intestinal mucosa and improved blood uptake of IND. Because of the reactive oxygen species-scavenging effect, IND@RNP(O) did not cause severe inflammation in the small intestine; this effect sharply contrasted with those of orally administered free-IND and IND-loaded polymeric micelles that do not possess reactive oxygen species scavengers. CONCLUSION: Oral IND@RNP(O) administration is a useful approach for improving the oral bioavailability of IND and suppressing its adverse effects.

DOI： 10.4155/tde.13.133

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Reactive oxygen species (ROS) scavengers have not been widely used for treatment of local inflammatory reactions such as arthritis and periodontal disease because they are rapidly eliminated from the inflamed site, which results in a low therapeutic effect. Therefore, to enhance the local retention time of ROS scavengers, we developed a redox-active injectable gel (RIG) system by using poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PMNT-PEG-PMNT) triblock copolymer, which possesses ROS scavenging nitroxide radicals as side chains of the PMNT segment. Cationic PMNT segment in PMNT-PEG-PMNT forms polyion complexes with anionic poly(acrylic acid) (PAAc) to form a flower-like micelle (ca. 79 nm), which exhibits in situ thermo-irreversible gelation under physiological conditions. We confirmed the prolonged site-specific retention time of RIG by performing in vivo noninvasive electron spin resonance imaging and quantitative evaluation. In contrast to low-molecular-weight nitroxide radical compounds that disappeared from injection sites in less than 1h after subcutaneous injection, 40% of the RIG remained even at 3 days. We also found that RIG inhibits neutrophil infiltration and cytokine production, which leads to suppression of hyperalgesia. These findings indicate the potential of RIG as an innovative approach for treatment of local inflammation.

DOI： 10.1016/j.jconrel.2013.10.009

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The ultimate goal of cancer chemotherapy is to achieve a cure without causing any adverse effects. We have developed a pH-sensitive redox nanoparticle (RNP(N)), which disintegrates under acidic conditions and exposes nitroxide radicals, leading to strongly scavenging reactive oxygen species (ROS). After intravenous administration of RNP(N) to tumor bearing mice, it effectively accumulated in tumors due to the leaky neovascular and immature lymphatic system and scavenged ROS, resulting in suppression of inflammation and activation of NF-кB, after disintegration of RNP(N) in the tumors. Pre-administration of RNP(N) prior to treatments with anticancer agents, doxorubicin, to tumor-bearing mice significantly suppressed the progression of tumor size, compared to low-molecular weight 4-hydroxy-TEMPO. Interestingly, the administration of RNP(N) suppressed adverse effects of doxorubicin to normal organs due to the scavenging ROS and suppression of inflammation, which was confirmed by reduction in lactate dehydrogenase and creatine phosphokinase activities in plasma. RNP(N) is thus anticipated as a novel and ideal adjuvant for cancer chemotherapy.

DOI： 10.1016/j.jconrel.2013.08.011

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Patients regularly taking non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin (IND) have a risk of small intestinal injuries. In this study, we have developed an oral nanotherapeutics by using a redox nanoparticle (RNP(O)), which is prepared by self-assembly of an amphiphilic block copolymer that possesses nitroxide radicals as side chains of hydrophobic segment via ether linkage, to reduce inflammation in mice with IND-induced small intestinal injury. The localization and accumulation of RNP(O) in the small intestine were determined using fluorescent-labeled RNP(O) and electron spin resonance. After oral administration, the accumulation of RNP(O) in both the jejunum and ileum tissues was about 40 times higher than those of low-molecular-weight nitroxide radical compounds, and RNP(O) was not absorbed into the bloodstream via the mesentery, thereby avoiding the adverse effects of nitroxide radicals in the entire body. RNP(O) remarkably suppressed inflammatory mediators such as myeloperoxidase, superoxide anion, and malondialdehyde in the small intestines of IND-treated mice. Compared to low-molecular-weight nitroxide radical compounds, RNP(O) also significantly increased the survival rate of mice treated daily with IND. On the basis of these results, RNP(O) is promising as a nanotherapeutics for treatment of inflammation in the small intestine of patients receiving NSAIDs.

DOI： 10.1016/j.biomaterials.2013.06.032

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To image inflammation sites, we developed a novel nanoparticle, hydroxylamine-containing nanoparticle (HANP), which emits an intense electron spin resonance (ESR)-signal triggered by enzymatic oxidation reaction and pH-sensitive self-disintegration. The nanoparticle was prepared from an amphiphilic block copolymer, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-hydroxyl)aminomethylstyrene] (PEG-b-PMNT-H), which spontaneously forms a core-shell type polymeric micelle (particle diameter = ca. 50 nm) in aqueous media. Because the PMNT-H segment in the block copolymer possesses amino groups in each repeating unit, the particle can be disintegrated by protonation of the amino groups in an acidic pH environment such as inflammation sites, which is confined to the hydrophobic core of HANP. Mixing HANP with horseradish peroxidase (HRP)/H(2)O(2) mixture resulted in enzymatic oxidization of the hydroxylamines in the PEG-b-PMNT-H and converted the hydroxylamine to the stable nitroxide radical form in PEG-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT), which shows an intense ESR signal. It is interesting to note that the ESR signal increased at a greater rate under acidic conditions (pH 5.6) than that under neutral conditions (pH 7.4), although the enzymatic activity of HRP under neutral conditions is known to be much higher than that under acidic conditions. This indicates that enzymatic oxidation reaction was accelerated by synchronizing the disintegration of HANP under acidic conditions. On the basis of these results, HANP can be used as a high-performance ESR probe for imaging of inflammation sites.

DOI： 10.1007/s13758-011-0007-5

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BACKGROUND & AIMS: Drugs used to treat patients with ulcerative colitis are not always effective because of nonspecific distribution, metabolism in the gastrointestinal tract, and side effects. We designed a nitroxide radical-containing nanoparticle (RNP(O)) that accumulates specifically in the colon to suppress inflammation and reduce the undesirable side effects of nitroxide radicals. METHODS: RNP(O) was synthesized by assembly of an amphiphilic block copolymer that contains stable nitroxide radicals in an ether-linked hydrophobic side chain. Biodistribution of RNP(O) in mice was determined from radioisotope and electron spin resonance measurements. The effects of RNP(O) were determined in mice with dextran sodium sulfate (DSS)-induced colitis and compared with those of low-molecular-weight drugs (4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl [TEMPOL] or mesalamine). RESULTS: RNP(O), with a diameter of 40 nm and a shell of poly(ethylene glycol), had a significantly greater level of accumulation in the colonic mucosa than low-molecular-weight TEMPOL or polystyrene latex particles. RNP(O) was not absorbed into the bloodstream through the intestinal wall, despite its long-term retention in the colon, which prevented its distribution to other parts of the body. Mice with DSS-induced colitis had significantly lower disease activity index and less inflammation following 7 days of oral administration of RNP(O) compared with mice with DSS-induced colitis or mice given low-molecular-weight TEMPOL or mesalamine. CONCLUSIONS: We designed an orally administered RNP(O) that accumulates specifically in the colons of mice with colitis and is more effective in reducing inflammation than low-molecular-weight TEMPOL or mesalamine. RNP(O) might be developed for treatment of patients with ulcerative colitis.

DOI： 10.1053/j.gastro.2012.06.043

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BACKGROUND: Intracerebral hemorrhage is reported to induce the generation of reactive oxygen species and oxidative DNA damage in the brain. AIMS: We aimed to examine whether our designed redox polymer nanoparticle could reduce intracerebral hemorrhage induced by 1-MHz focused ultrasound sonication coupled with microbubble treatment. MATERIALS & METHODS: Contrast-enhanced ultrasound imaging, frozen section, brain edema, neurologic deficit, the number of morphologically normal neurons, DNA oxidization and superoxide anion generation were used to investigate the neuroprotective effect of redox polymer nanoparticles. RESULTS: We confirmed that the 1-MHz focused ultrasound coupled with microbubble produced intracerebral hemorrhage and showed that the redox polymer nanoparticle ameliorates intracerebral hemorrhage-induced brain edema, neurological deficit and oxidative damage. CONCLUSION: These results suggest that redox polymer nanoparticle is a potential therapeutic agent for intracerebral hemorrhage induced by focused ultrasound.

DOI： 10.2217/nnm.12.2

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Various polymeric materials have been used in medical devices, including blood-contacting artificial organs. Contact between blood and foreign materials causes blood cell activation and adhesion, followed by blood coagulation. Concurrently, the activated blood cells release inflammatory cytokines together with reactive oxygen species (ROS). We have hypothesized that the suppression of ROS generation plays a crucial role in blood activation and coagulation. To confirm this hypothesis, surface-coated polymers containing nitroxide radical compounds (nitroxide radical-containing polymers (NRP)) were designed and developed. The NRP was composed of a hydrophobic poly(chloromethylstyrene) (PCMS) chain to which 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moieties were conjugated via condensation reaction of the chloromethyl groups in PCMS with the sodium alcoholate group of 4-hydroxy-TEMPO. Blood compatibility was investigated by placing NRP-coated beads in contact with rat whole blood. The amount of ROS generated on PCMS-coated beads used as a control increased significantly with time, while NRP-coated beads suppressed ROS generation. It is interesting to note that the suppression of inflammatory cytokine generation by NRP-coated beads was shown to be significantly higher than that by PCMS-coated beads. Both platelet and leukocyte adhesion to the beads were suppressed with increasing TEMPO incorporation in the polymer. These results confirm that the suppression of ROS by NRP prevents inflammatory cytokine generation, which in turn results in the suppression of blood activation and coagulation on the beads.

DOI： 10.1016/j.actbio.2011.11.029

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Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

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The ultimate objective of nanoparticle-based therapy is to functionalize nanomedicines in a micro-disease environment without any side effects. Here, we reveal that our pH-responsive nitroxide radical-containing nanoparticles (RNP(pH)) disintegrate within the renal acidic lesion and act as scavengers of reactive oxygen species (ROS), leading to a relief of acute kidney injury (AKI). RNP(pH) was prepared using amphiphilic block copolymers possessing 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moieties via amine linkage as a side chain of the hydrophobic segment. The self-assembled RNP(pH) disintegrated at pH below 7.0 because of a protonation of the amino groups in the hydrophobic core of the nanoparticles, thereby resulting in an improvement in ROS scavenging activity. Using a renal ischemia-reperfusion AKI model in mice, the therapeutic effect of RNP(pH) on ROS damage was evaluated. Unlike the RNP without pH-triggered disintegration (RNP(Non-pH)), the RNP(pH) showed extremely high ROS scavenging activity and renal protective effects. It is interesting to note that the side effect of nitroxide radicals was markedly suppressed due to the compartmentalization of nitroxide radicals in the core of RNP(pH) in untargeted area. The morphology changes in RNP(pH) were confirmed by analyzing electron spin resonance spectra, and these findings provide the evidence of the real therapeutic effect of the environment-sensitive specific disintegration of nanoparticles in vivo.

DOI： 10.1016/j.biomaterials.2011.07.014

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The antioxidant effect and potential mechanism of nitroxide radical-containing nanoparticles (RNPs) coupled with piperine (PI) were investigated in human neuroblastoma SH-SY5Y cells. The effects of RNP/PI on SH-SY5Y cell lines was determined by WST assay for cell viability, nitroblue tetrazolium and deoxyribose assay for reactive oxygen species generation, ELISA assay for reactive oxygen species products and apoptotic cell death, and biochemical techniques for catalase and glutathione peroxidase activity. The RNP/PI significantly reduced the reactive oxygen species level and reactive oxygen species products compared with those of cells treated with RNPs alone. The RNP/PI treatment enhanced catalase and glutathione peroxidase activity. The combination of RNP/PI has been found to have an augmented antioxidant effect on an Alzheimer's model in vitro. The mechanism of the protective effect of this combination therapy was correlated in this study with its ability to reduce the generation of reactive oxygen species and prevent apoptosis via scavenging enzyme action pathways.

DOI： 10.1016/j.biomaterials.2011.07.024

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BACKGROUND: Excessive accumulation of beta-amyloid (Abeta) has been proposed as a pivotal event in the pathogenesis of Alzheimer's disease. Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excess production of reactive oxygen species (ROS) and apoptosis. We have designed novel nanoparticles, nitroxyl radical-containing nanoparticles (RNPs), which possess nitroxyl radical in the core and chemically scavenges ROS. This study aimed to determine the potential neuroprotective role of RNPs on Abeta-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. METHOD: SH-SY5Y cells were preincubated with 0.1-1 mM RNP for 24 h and then incubated with 20 microM Abeta1-42, for 48 h. In every group, cell viability, apoptotic rate, ROS levels including superoxide anion radicals and hydroxyl radicals, ROS production including lipid peroxidation, protein oxidation and DNA oxidation were measured. RESULTS: SH-SY5Y cells preincubated with 0.1-2 mM RNP for 24 h were protected from Abeta-induced damage. SH-SY5Y cells preincubated with more than 2 mM RNPfor 24 h showed cytotoxicity. From the quantitative analyses, it was observed that RNPs reduced intracellular oxidative stress. RNP treatment significantly reduced the amount of oxidized lipids, proteins and DNA. It also reduced DNA fragmentations, which caused lower apoptosis levels. CONCLUSION: RNPs are promising intracellular ROS scavengers.

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BACKGROUND: Antioxidant nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) have been investigated for their ability to scavenge free radicals produced by ischemia-reperfusion injury. However, the short in vivo half-life and toxicity of TEMPO have limited their clinical application. OBJECTIVE: We developed a core-shell-type nanoparticle, termed a radical-containing nanoparticle (RNP), to deliver nitroxyl radicals with prolonged in vivo half-life and pH-sensitivity. We evaluated the ability of RNP to deliver TEMPO radicals to the ischemic brain and scavenge free radicals in cerebral ischemia-reperfusion injury using rats. METHODS: When RNPs were administrated to middle cerebral artery occlusion rats, the delivery and clearance of RNPs were detected using electron paramagnetic resonance (EPR) assay. The production of superoxide anion in neuronal cells was observed with dihydroethidium staining. The treatment effects were evaluated by measuring the cerebral infarction volumes, lipid peroxidation and protein oxidation, and neurological symptom scoring. RESULTS: The TEMPO radicals contained in RNPs were detected for 6 hours after intravenous administration as a triplet EPR signal in the ischemic brain, and RNPs significantly reduced the production of superoxide anion in neuronal cells compared with saline and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyls (TEMPOL). The infarction volumes of rats treated by RNPs were significantly lower than those of rats treated by saline, micelles, and TEMPOL. In addition, RNP treatment suppressed lipid peroxidation and protein oxidation, and limited the adverse effects of TEMPO radicals such as hypotension. CONCLUSION: RNPs could be a promising neuroprotective agent with their enhanced ability to scavenge free radicals and reduced toxicity.

DOI： 10.1227/NEU.0b013e31820c02d9

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This article discusses the preparation and characterization of pH-sensitive nitroxyl radical-containing nanoparticles (RNPs) possessing nitroxyl radicals in the core and reactive groups on the periphery, and its biomedical application. The RNPs prepared by a self-assembling amphiphilic block copolymers composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxyls (TEMPOs) via an amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO are initially described. The cumulant average diameter of an RNP is approximately 40 nm, and the RNP has intense electron paramagnetic resonance signals. RNPs show a prolonged blood circulation time by the compartmentalization of nitroxyl radicals into the hydrophobic core, and disintegrate in response to a low pH environment, such as ischemic tissue, resulting in effectively scavenging reactive oxygen species due to an exposure of nitroxyl radicals from the RNP core. Thus, the RNP prepared was found to be effective for cerebral ischemia-reperfusion injury. Therefore, RNPs are promising as high-performance therapeutic nanomedicine for oxidative stress injuries.

DOI： 10.2217/nnm.11.13

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A novel oligonucleotide carrier which can scavenge ROS is described. The synthesized graft polymer is composed of a PEG segment and a TEMPO-containing hydrophobic segment for scavenging ROS. This graft polymer can form a PIC through electrostatic interaction with oligonucleotides such as siRNA. The amount of ROS was monitored by fluorescence measurements using H₂ DCFDA as a probe, and it was confirmed that the ROS level was effectively suppressed. The cellular uptake of PIC containing the fluorescence-labeled oligonucleotide was evaluated by fluorescence microscopy. Delivered siRNA suppressed the expression of the mRNA. The prepared graft copolymer is thus a promising candidate as a novel oligonucleotide carrier which also reduces ROS damage generated by cationic polymer carriers.

DOI： 10.1002/mabi.201000305

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DOI： 10.1016/j.freeradbiomed.2010.10.429

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For the imaging of low pH circumstances in vivo, a pH-sensitive radical-containing-nanoparticle (RNP), which has an intense electron paramagnetic resonance (EPR) signal, was designed and developed using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO. This RNP formed core-shell-type micelles in the physiological environment, and the cumulant average diameter of the RNP was about 50 nm. The cytotoxicity and acute toxicity studies for the RNP revealed that the median inhibitory concentration (IC(50)) of TEMPO radicals in RNP core and median lethal dose (LD(50)) of RNP were >8 mmol N(TEMPO)/L and >600 mg/kg (>960 mumol N(TEMPO)/kg), respectively, indicating fairly low toxicity. The blood circulation of the RNP was evaluated using ICR mice. Contrary to the rapid clearance of low-molecular-weight TEMPO derivatives such as 4-hydroxy-TEMPO (TEMPOL) from the bloodstream, the EPR signal of the RNP remained for a fairly long period of time. Actually, the signal was observed in the blood for more than 2 h, as monitored by EPR spectroscopy. The compartmentalization of the TEMPO radicals in the RNP core improved the stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PCTEMPO segment, the disintegration of the RNP was caused by the protonation of the amino groups in response to the acidic pH environment (pH < 6.0), as confirmed by the dynamic light scattering (DLS) measurements. In addition, a drastic change in the EPR spectra from broad to sharp triplet was observed, accompanying the disintegration. This change was based upon the mobility of the TEMPO moieties covalently conjugated in the hydrophobic segment, which was confirmed by the rotational correlation time of the TEMPO moieties on the PCTEMPO segment. Note that the peak intensity of the EPR signal increased at around the phase transition point (ca. pH = 6.0). When pH-sensitive RNP solutions at pH values 5.6 and 7.4 were visualized using an L-band EPR imaging system, the phantom images showed a remarkable on-off regulation in response to the acidic pH environment. These results demonstrate that pH-sensitive RNPs are expected to serve as nanoprobes for the in vivo EPR imaging of low pH circumstances.

DOI： 10.1021/bc900214f

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Utilizing the self-assembled core-shell-type polymeric micelle technique, high-performance nanoparticles possessing stable radicals in the core and reactive groups on the periphery were prepared. The anionic ring-opening polymerization of ethylene oxide (EO) was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)-methanesulfonate (acetal-PEG-Ms; 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl and acetal terminal groups (acetal-PEG-SH) (2) in a highly selective and quantitative manner. Poly(ethylene glycol)-block-poly(chloromethylstyrene) (acetal-PEG-b-PCMS) (3) was synthesized by the free-radical telomerization of chloromethylstyrene (CMS) using 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (4). The obtained 4 formed core-shell-type nanoparticles in aqueous media when subjected to the dialysis method: the cumulant average diameter of the nanoparticles was about 40 nm, and the nanoparticles emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid. This means that these nanoparticles are anticipated as high-performance bionanoparticles that can be used in vivo.

DOI： 10.1021/bm801278n

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