Anticancer Activity of Astaxanthin-Incorporated Chitosan Nanoparticles.

Astaxanthin (AST)-encapsulated nanoparticles were fabricated using glycol chitosan (Chito) through electrostatic interaction (abbreviated as ChitoAST) to solve the aqueous solubility of astaxanthin and improve its biological activity. AST was dissolved in organic solvents and then mixed with chitosan solution, followed by a dialysis procedure. All formulations of ChitoAST nanoparticles showed small diameters (less than 400 nm) with monomodal distributions. Analysis with Fourier transform infrared (FT-IR) spectroscopy confirmed the specific peaks of AST and Chito. Furthermore, ChitoAST nanoparticles were formed through electrostatic interactions between Chito and AST. In addition, ChitoAST nanoparticles showed superior antioxidant activity, as good as AST itself; the half maximal radical scavenging concentrations (RC50) of AST and ChitoAST nanoparticles were 11.8 and 29.3 µg/mL, respectively. In vitro, AST and ChitoAST nanoparticles at 10 and 20 µg/mL properly inhibited the production of intracellular reactive oxygen species (ROSs), nitric oxide (NO), and inducible nitric oxide synthase (iNOS). ChitoAST nanoparticles had no significant cytotoxicity against RAW264.7 cells or B16F10 melanoma cells, whereas AST and ChitoAST nanoparticles inhibited the growth of cancer cells. Furthermore, AST itself and ChitoAST nanoparticles (20 µg/mL) efficiently inhibited the migration of cancer cells in a wound healing assay. An in vivo study using mice and a pulmonary metastasis model showed that ChitoAST nanoparticles were efficiently delivered to a lung with B16F10 cell metastasis; i.e., fluorescence intensity in the lung was significantly higher than in other organs. We suggest that ChitoAST nanoparticles are promising candidates for antioxidative and anticancer therapies of B16F10 cells.

ß-Cyclodextrin Nanophotosensitizers for Redox-Sensitive Delivery of Chlorin e6.

The aim of this study is to prepare redox-sensitive nanophotosensitizers for the targeted delivery of chlorin e6 (Ce6) against cervical cancer. For this purpose, Ce6 was conjugated with ß-cyclodextrin (bCD) via a disulfide bond, creating nanophotosensitizers that were fabricated for the redox-sensitive delivery of Ce6 against cancer cells. bCD was treated with succinic anhydride to synthesize succinylated bCD (bCDsu). After that, cystamine was attached to the carboxylic end of bCDsu (bCDsu-ss), and the amine end group of bCDsu-ss was conjugated with Ce6 (bCDsu-ss-Ce6). The chemical composition of bCDsu-ss-Ce6 was confirmed with 1H and 13C NMR spectra. bCDsu-ss-Ce6 nanophotosensitizers were fabricated by a dialysis procedure. They formed small particles with an average particle size of 152.0 ± 23.2 nm. The Ce6 release rate from the bCDsu-ss-Ce6 nanophotosensitizers was accelerated by the addition of glutathione (GSH), indicating that the bCDsu-ss-Ce6 nanophotosensitizers have a redox-sensitive photosensitizer delivery capacity. The bCDsu-ss-Ce6 nanophotosensitizers have a low intrinsic cytotoxicity against CCD986Sk human skin fibroblast cells as well as Ce6 alone. However, the bCDsu-ss-Ce6 nanophotosensitizers showed an improved Ce6 uptake ratio, higher reactive oxygen species (ROS) production, and phototoxicity compared to those of Ce6 alone. GSH addition resulted in a higher Ce6 uptake ratio, ROS generation, and phototoxicity than Ce6 alone, indicating that the bCDsu-ss-Ce6 nanophotosensitizers have a redox-sensitive biological activity in vitro against HeLa human cervical cancer cells. In a tumor xenograft model using HeLa cells, the bCDsu-ss-Ce6 nanophotosensitizers efficiently accumulated in the tumor rather than in normal organs. In other words, the fluorescence intensity in tumor tissues was significantly higher than that of other organs, while Ce6 alone did not specifically target tumor tissue. These results indicated a higher anticancer activity of bCDsu-ss-Ce6 nanophotosensitizers, as demonstrated by their efficient inhibition of the growth of tumors in an in vivo animal tumor xenograft study.

Biological Synthesis of Copper Nanoparticles Using Edible Plant Allium monanthum: Characterization of Antibacterial, Antioxidant, and Anti-Inflammatory Properties Using In Silico Molecular Docking Analysis.

This study prepared copper nanoparticles using an edible leaf extract from A. monanthum (AM-CuNPs) via eco-friendly green synthesis techniques. The size, shape, crystalline nature and functional groups of the synthesized AM-CuNP particles were analyzed by a UV-VIS spectrophotometer and SEM, EDX, TEM, XRD and FT-IR instrumentation. The synthesized AM-CuNPs had spherical shapes with sizes in the range of 30-80 nm and were crystalline in nature. In addition, the AM-CuNPs were synthesized using various bioactive sources, including flavonoids, phenolic acids, alkaloids and sugars that were present in an aqueous broth of A. monanthum. Furthermore, the AM-CuNPs possessed good antibacterial properties against selected major disease-causing pathogenic bacteria, such as E. coli, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus. The antioxidant activity of AM-CuNPs exhibited potent free radical scavenging activities in DPPH, ABTS and H2O2 radical assays. In addition, in silico analysis of the AM-CuNPs was performed, including ADME prediction, and molecular simulation docking on the secondary metabolites identified in the edible plant extract was used to evaluate their anti-inflammatory applications. In particular, the molecular docking scores showed that alliin, apigenin, isorhamnetin, luteolin and myricetin have sufficient binding energy and top values as inhibitors of the protein target involved in the inflammation signaling cascade.

Synergistic Effects of Radiotherapy With JNK Inhibitor-Incorporated Nanoparticle in an Intracranial Lewis Lung Carcinoma Mouse Models.

BACKGROUND: Radiosurgery has been recognized as a reasonable treatment for metastatic brain tumors. Increasing the radiosensitivity and synergistic effects are possible ways to improve the therapeutic efficacy of specific regions of tumors. c-Jun-N-terminal kinase (JNK) signaling regulates H2AX phosphorylation to repair radiation-induced DNA breakage. We previously showed that blocking JNK signaling influenced radiosensitivity in vitro and in an in vivo mouse tumor model. Drugs can be incorporated into nanoparticles to produce a slow-release effect. This study assessed JNK radiosensitivity following the slow release of the JNK inhibitor SP600125 from a poly (DL-lactide-co-glycolide) (LGEsese) block copolymer in a brain tumor model. MATERIALS AND METHODS: A LGEsese block copolymer was synthesized to fabricate SP600125-incorporated nanoparticles by nanoprecipitation and dialysis methods. The chemical structure of the LGEsese block copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The physicochemical and morphological properties were observed by transmission electron microscopy (TEM) imaging and measured with particle size analyzer. The blood-brain barrier (BBB) permeability to the JNK inhibitor was estimated by BBBflammaTM 440-dye-labeled SP600125. The effects of the JNK inhibitor were investigated using SP600125-incorporated nanoparticles and by optical bioluminescence, magnetic resonance imaging (MRI), and a survival assay in a mouse brain tumor model for Lewis lung cancer (LLC)-Fluc cells. DNA damage was estimated by histone γ H2AX expression and apoptosis was assessed by the immunohistochemical examination of cleaved caspase 3. RESULTS: The SP600125-incorporated nanoparticles of the LGEsese block copolymer were spherical and released SP600125 continuously for 24h. The use of BBBflammaTM 440-dye-labeled SP600125 demonstrated the ability of SP600125 to cross the BBB. The blockade of JNK signaling with SP600125-incorporated nanoparticles significantly delayed mouse brain tumor growth and prolonged mouse survival after radiotherapy. γ H2AX, which mediates DNA repair protein, was reduced and the apoptotic protein cleaved-caspase 3 was increased by the combination of radiation and SP600125-incorporated nanoparticles.

Phenethyl Isothiocyanate-Conjugated Chitosan Oligosaccharide Nanophotosensitizers for Photodynamic Treatment of Human Cancer Cells.

The aim of this study is to synthesize phenethyl-conjugated chitosan oligosaccharide (COS) (abbreviated as ChitoPEITC) conjugates and then fabricate chlorin E6 (Ce6)-incorporated nanophotosensitizers for photodynamic therapy (PDT) of HCT-116 colon carcinoma cells. PEITC was conjugated with the amine group of COS. Ce6-incorporated nanophotosensitizers using ChitoPEITC (ChitoPEITC nanophotosensitizers) were fabricated by dialysis method. 1H nuclear magnetic resonance (NMR) spectra showed that specific peaks of COS and PEITC were observed at ChitoPEITC conjugates. Transmission electron microscope (TEM) confirmed that ChitoPEITC nanophotosensitizers have spherical shapes with small hydrodynamic diameters less than 200 nm. The higher PEITC contents in the ChitoPEITC copolymer resulted in a slower release rate of Ce6 from nanophotosensitizers. Furthermore, the higher Ce6 contents resulted in a slower release rate of Ce6. In cell culture study, ChitoPEITC nanophotosensitizers showed low toxicity against normal CCD986Sk human skin fibroblast cells and HCT-116 human colon carcinoma cells in the absence of light irradiation. ChitoPEITC nanophotosensitizers showed a significantly higher Ce6 uptake ratio than that of free Ce6. Under light irradiation, cellular reactive oxygen species (ROS) production of nanophotosensitizers was significantly higher than that of free Ce6. Especially, PEITC and/or ChitoPEITC themselves contributed to the production of cellular ROS regardless of light irradiation. ChitoPEITC nanophotosensitizers showed significantly higher PDT efficacy against HCT-116 cells than that of free Ce6. These results indicate that ChitoPEITC nanophotosensitizers have superior potential in Ce6 uptake, ROS production and PDT efficacy. In the HCT-116 cell-bearing mice tumor-xenograft model, ChitoPEITC nanophotosensitizers efficiently inhibited growth of tumor volume rather than free Ce6. In the animal imaging study, ChitoPEITC nanophotosensitizers were concentrated in the tumor tissue, i.e., fluorescence intensity in the tumor tissue was stronger than that of other tissues. We suggest that ChitoPEITC nanophotosensitizers are a promising candidate for the treatment of human colon cancer cells.

Nanophotosensitizers Composed of Phenyl Boronic Acid Pinacol Ester-Conjugated Chitosan Oligosaccharide via Thioketal Linker for Reactive Oxygen Species-Sensitive Delivery of Chlorin e6 against Oral Cancer Cells.

Chlorin E6 (Ce6)-incorporated nanophotosensitizers were fabricated for application in photodynamic therapy (PDT) of oral cancer cells. For this purpose, chitosan oligosaccharide (COS) was conjugated with hydrophobic and reactive oxygen species (ROS)-sensitive moieties, such as phenyl boronic acid pinacol ester (PBAP) via a thioketal linker (COSthPBAP). ThdCOOH was conjugated with PBAP to produce ThdCOOH-PBAP conjugates and then attached to amine groups of COS to produce a COSthPBAP copolymer. Ce6-incorporated nanophotosensitizers using the COSthPBAP copolymer were fabricated through the nanoprecipitation and dialysis methods. The Ce6-incorporated COSthPBAP nanophotosensitizers had a small diameter of less than 200 nm with a mono-modal distribution pattern. However, it became a multimodal and/or irregular distribution pattern when H2O2 was added. In a morphological observation using TEM, the nanophotosensitizers were disintegrated by the addition of H2O2, indicating that the COSthPBAP nanophotosensitizers had ROS sensitivity. In addition, the Ce6 release rate from the COSthPBAP nanophotosensitizers accelerated in the presence of H2O2. The SO generation was also higher in the nanophotosensitizers than in the free Ce6. Furthermore, the COSthPBAP nanophotosensitizers showed a higher intracellular Ce6 uptake ratio and ROS generation in all types of oral cancer cells. They efficiently inhibited the viability of oral cancer cells under light irradiation, but they did not significantly affect the viability of either normal cells or cancer cells in the absence of light irradiation. The COSthPBAP nanophotosensitizers showed a tumor-specific delivery capacity and fluorescence imaging of KB tumors in an in vivo animal tumor imaging study. We suggest that COSthPBAP nanophotosensitizers are promising candidates for the imaging and treatment of oral cancers.

Antibacterial Activity of Antibiotic-Releasing Polydopamine-Coated Nephrite Composites for Application in Drug-Eluting Contact Lens.

The aim of this study is to prepare ciprofloxacin (CIP) or levofloxacin (LEVO)-incorporated and polydopamine (PDA)-coated nephrite composites for application in drug-eluting contact lenses. PDA was coated onto the surface of nephrite to improve antibacterial activity and to payload antibiotics. CIP or LEVO was incorporated into the PDA layer on the surface of nephrite. Furthermore, CIP-incorporated/PDA-coated nephrite composites were embedded into the contact lenses. PDA-coated nephrite composites showed dull and smooth surfaces according to the dopamine concentration while nephrite itself has sharp surface morphology. CIP- or LEVO-loaded/PDA-coated nephrite composites also have dull and smooth surface properties. Nano and/or sub-micron clusters were observed in field emission-scanning electron microscopy (FE-SEM) observation, indicating that PDA nanoparticles were accumulated and coated onto the surface of nephrite. Furthermore, CIP- or LEVO-incorporated/PDA-coated nephrite composites showed the sustained release of CIP or LEVO in vitro and these properties contributed to the enhanced antibacterial activity of composites compared to nephrite or PDA-coated nephrite composites. CIP-incorporated/PDA-coated nephrite composites were embedded in the contact lenses and then, in an antibacterial study, they showed higher bactericidal effect against Staphylococcus aureus (S. aureus) compared to nephrite itself or PDA-coated nephrite composites. We suggest that CIP- or LEVO-loaded/PDA-coated nephrite composite-embedded contact lenses are a promising candidate for therapeutic application.

pH and Redox-Dual Sensitive Chitosan Nanoparticles Having Methyl Ester and Disulfide Linkages for Drug Targeting against Cholangiocarcinoma Cells.

The aim of this study is to prepare pH- and redox-sensitive nanoparticles for doxorubicin (DOX) delivery against DOX-resistant HuCC-T1 human cholangiocarcinoma (CCA) cells. For this purpose, L-histidine methyl ester (HIS) was attached to chitosan oligosaccharide (COS) via dithiodipropionic acid (abbreviated as ChitoHISss). DOX-incorporated nanoparticles of ChitoHISss conjugates were fabricated by a dialysis procedure. DOX-resistant HuCC-T1 cells were prepared by repetitive exposure of HuCC-T1 cells to DOX. ChitoHISss nanoparticles showed spherical morphology with a small diameter of less than 200 nm. The acid pH and glutathione (GSH) addition induced changes in the size distribution pattern of ChitoHISss nanoparticles from a narrow/monomodal distribution pattern to a wide/multimodal pattern and increased the fluorescence intensity of the nanoparticle solution. These results indicate that a physicochemical transition of nanoparticles can occur in an acidic pH or redox state. The more acidic the pH or the higher the GSH concentration the higher the drug release rate was, indicating that an acidic environment or higher redox states accelerated drug release from ChitoHISss nanoparticles. Whereas free DOX showed decreased anticancer activity at DOX-resistant HuCC-T1 cells, DOX-incorporated ChitoHISss nanoparticles showed dose-dependent anticancer activity. Intracellular delivery of DOX-incorporated ChitoHISss nanoparticles was relatively increased at an acidic pH and in the presence of GSH, indicating that DOX-incorporated ChitoHISss nanoparticles have superior acidic pH- and redox-sensitive behavior. In an in vivo tumor xenograft model, DOX-incorporated ChitoHISss nanoparticles were specifically delivered to tumor tissues and then efficiently inhibited tumor growth. We suggest that ChitoHISss nanoparticles are a promising candidate for treatment of CCA.

Biomedical Membrane of Fish Collagen/Gellan Gum Containing Bone Graft Materials.

The development of a guided bone regeneration (GBR) membrane with non-mammalian fish collagen has the advantage of low risk for transmission of infectious diseases in tissue regeneration. In this work, a fish collagen/gellan gum and bone graft material (FC/GG-BGM) composite GBR membrane were fabricated through solution blending and casting procedures in a vacuum. The membranes were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy observation (SEM), and atomic force microscope (AFM) analyses. FT-IR results suggested that ionic interactions were formed between FC and GG both in composite powder and membranes. In vivo experiments showed that these FC/GG-BGM composite membranes could generate osteoblast minerals and promote loose bone calcification, thus accelerating bone regeneration. At 2 weeks, the defected site of rats treated with FC/GG-BGM membrane (0.377 ± 0.012 mm3) showed higher regeneration than that of rats treated with the bovine collagen membrane (0.290 ± 0.015 mm3) and control rats without membrane (0.160 ± 0.008 mm3). Compared with bovine collagen membrane, the FC/GG-BGM composite membrane displays better bone regeneration ability. Therefore, FC/GG-BGM composite membrane is suitable as a GBR membrane for bone regeneration.

CD44 Receptor-Mediated/Reactive Oxygen Species-Sensitive Delivery of Nanophotosensitizers against Cervical Cancer Cells.

Stimulus-sensitive, nanomedicine-based photosensitizer delivery has an opportunity to target tumor tissues since oxidative stress and the expression of molecular proteins, such as CD44 receptors, are elevated in the tumor microenvironment. The aim of this study is to investigate the CD44 receptor- and reactive oxygen species (ROS)-sensitive delivery of nanophotosensitizers of chlorin e6 (Ce6)-conjugated hyaluronic acid (HA) against HeLa human cervical cancer cells. For the synthesis of nanophotosensitizers, thioketal diamine was conjugated with the carboxyl group in HA and then the amine end group of HA-thioketal amine conjugates was conjugated again with Ce6 (Abbreviated as HAthCe6). The HAthCe6 nanophotosensitizers were of small diameter, with sizes less than 200. Their morphology was round-shaped in the observations using a transmission electron microscope (TEM). The HAthCe6 nanophotosensitizers responded to oxidative stress-induced changes in size distribution when H2O2 was added to the nanophotosensitizer aqueous solution, i.e., their monomodal distribution pattern at 0 mM H2O2 was changed to dual- and/or multi-modal distribution patterns at higher concentrations of H2O2. Furthermore, the oxidative stress induced by the H2O2 addition contributed to the disintegration of HAthCe6 nanophotosensitizers in morphology, and this phenomenon accelerated the release rate of Ce6 from nanophotosensitizers. In a cell culture study using HeLa cells, nanophotosensitizers increased Ce6 uptake ratio, ROS generation and PDT efficacy compared to free Ce6. Since HA specifically bonds with the CD44 receptor of cancer cells, the pretreatment of free HA against HeLa cells decreased the Ce6 uptake ratio, ROS generation and PDT efficacy of HAthCe6 nanophotosensitizers. These results indicated that intracellular delivery of HAthCe6 nanophotosensitizers can be controlled by the CD44 receptor-mediated pathway. Furthermore, these phenomena induced CD44 receptor-controllable ROS generation and PDT efficacy by HAthCe6 nanophotosensitizers. During in vivo tumor imaging using HeLa cells, nanophotosensitizer administration showed that the fluorescence intensity of tumor tissues was relatively higher than that of other organs. When free HA was pretreated, the fluorescence intensity of tumor tissue was relatively lower than those of other organs, indicating that HAthCe6 nanophotosensitizers have CD44 receptor sensitivity and that they can be delivered by receptor-specific manner. We suggest that HAthCe6 nanophotosensitizers are promising candidates for PDT in cervical cancer.

Reactive Oxygen Species and Folate Receptor-Targeted Nanophotosensitizers Composed of Folic Acid-Conjugated and Poly(ethylene glycol)-Chlorin e6 Tetramer Having Diselenide Linkages for Targeted Photodynamic Treatment of Cancer Cells.

Folic acid-conjugated nanophotosensitizers composed of folic acid (FA), poly(ethylene glycol) (PEG) and chlorin e6 (Ce6) tetramer were synthesized using diselenide linkages for reactive oxygen species (ROS)- and folate receptor-specific delivery of photosensitizers. Ce6 was conjugated with 3-[3-(2-carboxyethoxy)-2,2-bis(2-carboxyethoxymethyl)propoxy]propanoic acid (tetra acid, or TA) to make Ce6 tetramer via selenocystamine linkages (TA-sese-Ce6 conjugates). In the carboxylic acid end group of the TA-sese-Ce6 conjugates, FA-PEG was attached again using selenocystamine linkages to make FA-PEG/TA-sese-Ce6 conjugates (abbreviated as FAPEGtaCe6 conjugates). Nanophotosensitizers were fabricated by a dialysis procedure. In the morphological observations, they showed spherical shapes with small diameters of less than 200 nm. Stability of the aqueous FAPEGtaCe6 nanophotosensitizer solution was maintained (i.e., their particle sizes were not significantly changed until 7 days later). When H2O2 was added to the nanophotosensitizer solution, the particle size distribution was changed from a monomodal pattern to a multimodal pattern. In addition, the fluorescence intensity and Ce6 release rate from the nanophotosensitizers were also increased by the addition of H2O2. These results indicated that the nanophotosensitizers had ROS-sensitive properties. In an in vitro cell culture study, an FAPEGtaCe6 nanophotosensitizer treatment against cancer cells increased the Ce6 uptake ratio, ROS generation and light-irradiated cytotoxicity (phototoxicity) compared with Ce6 alone against various cancer cells. When the folic acid was pretreated to block the folate receptors of the Y79 cells and KB cells (folate receptor-overexpressing cells), the intracellular Ce6 uptake, ROS generation and thereby phototoxicity were decreased, while the MCF-7 cells did not significantly respond to blocking of the folate receptors. These results indicated that they could be delivered by a folate receptor-mediated pathway. Furthermore, an in vivo pulmonary metastasis model using Y79 cells showed folate receptor-specific delivery of FAPEGtaCe6 nanophotosensitizers. When folic acid was pre-administered, the fluorescence intensity of the lungs was significantly decreased, indicating that the FAPEGtaCe6 nanophotosensitizers had folate receptor specificity in vitro and in vivo. We suggest that FAPEGtaCe6 nanophotosensitizers are promising candidates for a targeted photodynamic therapy (PDT) approach against cancer cells.

Stimuli-Responsive Drug Delivery of Doxorubicin Using Magnetic Nanoparticle Conjugated Poly(ethylene glycol)-g-Chitosan Copolymer.

Stimuli-responsive nanoparticles are regarded as an ideal candidate for anticancer drug targeting. We synthesized glutathione (GSH) and magnetic-sensitive nanocomposites for a dual-targeting strategy. To achieve this goal, methoxy poly (ethylene glycol) (MePEG) was grafted to water-soluble chitosan (abbreviated as ChitoPEG). Then doxorubicin (DOX) was conjugated to the backbone of chitosan via disulfide linkage. Iron oxide (IO) magnetic nanoparticles were also conjugated to the backbone of chitosan to provide magnetic sensitivity. In morphological observation, images from a transmission electron microscope (TEM) showed that IO nanoparticles were embedded in the ChitoPEG/DOX/IO nanocomposites. In a drug release study, GSH addition accelerated DOX release rate from nanocomposites, indicating that nanocomposites have redox-responsiveness. Furthermore, external magnetic stimulus concentrated nanocomposites in the magnetic field and then provided efficient internalization of nanocomposites into cancer cells in cell culture experiments. In an animal study with CT26 cell-bearing mice, nanocomposites showed superior magnetic sensitivity and then preferentially targeted tumor tissues in the field of external magnetic stimulus. Nanocomposites composed of ChitoPEG/DOX/IO nanoparticle conjugates have excellent anticancer drug targeting properties.

The Effect of Oxidative Stress and Memantine-Incorporated Reactive Oxygen Species-Sensitive Nanoparticles on the Expression of N-Methyl-d-aspartate Receptor Subunit 1 in Brain Cancer Cells for Alzheimer's Disease Application.

The aim of this study is to fabricate reactive oxygen species (ROS)-sensitive nanoparticles composed of succinyl ß-cyclodextrin (bCDsu), memantine and thioketal linkages for application in Alzheimer's disease, and to investigate the suppression of N-methyl-d-aspartate (NMDA) receptor 1 (NMDAR1) in cells. Thioketal diamine was attached to the carboxyl group of bCDsu to produce thioketal-decorated bCDsu conjugates (bCDsu-thioketal conjugates) and memantine was conjugated with thioketal dicarboxylic acid (memantine-thioketal carboxylic acid conjugates). Memantine-thioketal carboxylic acid conjugates were attached to bCDsu-thioketal conjugates to produce bCDsu-thioketal-memantine (bCDsuMema) conjugates. SH-SY5Y neuroblastoma cells and U87MG cells were used for NMDAR1 protein expression and cellular oxidative stress. Nanoparticles of bCDsuMema conjugates were prepared by means of a dialysis procedure. Nanoparticles of bCDsuMema conjugates had small particle sizes less than 100 nm and their morphology was found to be spherical in transmission electron microscopy observations (TEM). Nanoparticles of bCDsuMema conjugates responded to H2O2 and disintegrated or swelled in aqueous solution. Then, the nanoparticles rapidly released memantine according to the concentration of H2O2. In an in vivo animal imaging study, thioketal-decorated nanoparticles labelled with fluorescent dye such as chlorin e6 (Ce6) showed that the fluorescence intensity was stronger in the brain than in other organs, indicating that bCDsuMema nanoparticles can efficiently target the brain. When cells were exposed to H2O2, the viability of cells was time-dependently decreased. Memantine or bCDsuMema nanoparticles did not practically affect the viability of the cells. Furthermore, a western blot assay showed that the oxidative stress produced in cells using H2O2 increased the expression of NMDAR1 protein in both SH-SY5Y and U87MG cells. Memantine or bCDsuMema nanoparticles efficiently suppressed the NMDAR1 protein, which is deeply associated with Alzheimer's disease. Fluorescence microscopy also showed that H2O2 treatment induced green fluorescence intensity, which represents intracellular ROS levels. Furthermore, H2O2 treatment increased the red fluorescence intensity, which represents the NMDAR1 protein, i.e., oxidative stress increases the expression of NMDAR1 protein level in both SH-SY5Y and U87MG cells. When memantine or bCDsuMema nanoparticles were treated in cells, the oxidative stress-mediated expression of NMDAR1 protein in cells was significantly decreased, indicating that bCDsuMema nanoparticles have the capacity to suppress NMDAR1 expression in brain cells, which has relevance in terms of applications in Alzheimer's disease.

Redox-Sensitive and Folate-Receptor-Mediated Targeting of Cervical Cancer Cells for Photodynamic Therapy Using Nanophotosensitizers Composed of Chlorin e6-Conjugated ß-Cyclodextrin via Diselenide Linkage.

The aim of this study was to fabricate a reactive oxygen species (ROS)-sensitive and folate-receptor-targeted nanophotosensitizer for the efficient photodynamic therapy (PDT) of cervical carcinoma cells. Chlorin e6 (Ce6) as a model photosensitizer was conjugated with succinyl ß-cyclodextrin via selenocystamine linkages. Folic acid (FA)-poly(ethylene glycol) (PEG) (FA-PEG) conjugates were attached to these conjugates and then FA-PEG-succinyl ß-cyclodextrin-selenocystamine-Ce6 (FAPEGbCDseseCe6) conjugates were synthesized. Nanophotosensitizers of FaPEGbCDseseCe6 conjugates were fabricated using dialysis membrane. Nanophotosensitizers showed spherical shapes with small particle sizes. They were disintegrated in the presence of hydrogen peroxide (H2O2) and particle size distribution changed from monomodal distribution pattern to multimodal pattern. The fluorescence intensity and Ce6 release rate also increased due to the increase in H2O2 concentration, indicating that the nanophotosensitizers displayed ROS sensitivity. The Ce6 uptake ratio, ROS generation and cell cytotoxicity of the nanophotosensitizers were significantly higher than those of the Ce6 itself against HeLa cells in vitro. Furthermore, the nanophotosensitizers showed folate-receptor-specific delivery capacity and phototoxicity. The intracellular delivery of nanophotosensitizers was inhibited by folate receptor blocking, indicating that they have folate-receptor specificity in vitro and in vivo. Nanophotosensitizers showed higher efficiency in inhibition of tumor growth of HeLa cells in vivo compared to Ce6 alone. These results show that nanophotosensitizers of FaPEGbCDseseCe6 conjugates are promising candidates as PDT of cervical cancer.

Ciprofloxacin-Releasing ROS-Sensitive Nanoparticles Composed of Poly(Ethylene Glycol)/Poly(D,L-lactide-co-glycolide) for Antibacterial Treatment.

Since urinary tract infections (UTIs) are closely associated with oxidative stress, we developed ROS-sensitive nanoparticles for ciprofloxacin (CIP) delivery for inhibition of UTI. Poly(D,L-lactide-co-glycolide) (PLGA)- selenocystamine (PLGA-selenocystamine) conjugates were attached to methoxypoly(ethylene glycol) (PEG) tetraacid (TA) (TA-PEG) conjugates to produce a copolymer (abbreviated as LGseseTAPEG). Selenocystamine linkages were introduced between PLGA and TA to endow reactive oxygen species (ROS) sensitivity to nanoparticles. CIP-incorporated nanoparticles of LGseseTAPEG copolymer were fabricated by W/O/W/W emulsion method. CIP-incorporated nanoparticles responded to H2O2 and then their morphologies were disintegrated by incubation with H2O2. Furthermore, particle size distribution of nanoparticles was changed from mono-modal distribution pattern to multi-modal distribution pattern by addition of H2O2. CIP release from nanoparticles of LGseseTAPEG copolymer was faster in the presence of H2O2 than in the absence of it. In antibacterial study using Escherichia coli (E. coli), free CIP and free CIP plus empty nanoparticles showed dose-dependent inhibitory effect against growth of bacteria while CIP-incorporated nanoparticles have less antibacterial activity compared to free CIP. These results were due to that CIP-incorporated nanoparticles have sustained release properties. When free CIP or CIP-incorporated nanoparticles were introduced into dialysis membrane to mimic in vivo situation, CIP-incorporated nanoparticles showed superior antibacterial activity compared to free CIP. At cell viability assay, nanoparticles of LGseseTAPEG copolymer have no acute cytotoxicity against L929 mouse fibroblast cells and CCD986sk human skin fibroblast cells. We suggest LGseseTAPEG nanoparticles are a promising candidate for CIP delivery.

Caffeic Acid Phenethyl Ester-Incorporated Radio-Sensitive Nanoparticles of Phenylboronic Acid Pinacol Ester-Conjugated Hyaluronic Acid for Application in Radioprotection.

We synthesized phenylboronic acid pinacol ester (PBPE)-conjugated hyaluronic acid (HA) via thiobis(ethylamine) (TbEA) linkage (abbreviated as HAsPBPE conjugates) to fabricate the radiosensitive delivery of caffeic acid phenetyl ester (CAPE) and for application in radioprotection. PBPE was primarily conjugated with TbEA and then PBPE-TbEA conjugates were conjugated again with hyaluronic acid using carbodiimide chemistry. CAPE-incorporated nanoparticles of HAsPBPE were fabricated by the nanoprecipitation method and then the organic solvent was removed by dialysis. CAPE-incorporated HAsPBPE nanoparticles have a small particle size of about 80 or 100 nm and they have a spherical shape. When CAPE-incorporated HAsPBPE nanoparticles were irradiated, nanoparticles became swelled or disintegrated and their morphologies were changed. Furthermore, the CAPE release rate from HAsPBPE nanoparticles were increased according to the radiation dose, indicating that CAPE-incorporated HAsPBPE nanoparticles have radio-sensitivity. CAPE and CAPE-incorporated HAsPBPE nanoparticles appropriately prevented radiation-induced cell death and suppressed intracellular accumulation of reactive oxygen species (ROS). CAPE and CAPE-incorporated HAsPBPE nanoparticles efficiently improved survivability of mice from radiation-induced death and reduced apoptotic cell death. We suggest that HAsPBPE nanoparticles are promising candidates for the radio-sensitive delivery of CAPE.

Reactive Oxygen Species-Sensitive Nanophotosensitizers of Methoxy Poly(ethylene glycol)-Chlorin e6/Phenyl Boronic Acid Pinacol Ester Conjugates Having Diselenide Linkages for Photodynamic Therapy of Cervical Cancer Cells.

The aim of this study is to fabricate nanophotosensitizers composed of methoxy poly(ethylene glycol) (mPEG), chlorin e6 (Ce6), and phenylboronic acid pinacol ester (PBAP) with diselenide linkages for reactive oxygen species (ROS)-sensitive photodynamic therapy (PDT) of cervical cancer cells. To fabricate nanophotosensitizers, Ce6 was conjugated with mPEG via selenocystamine linkage and then remaining carboxylic acid groups of Ce6 was attached to PBAP (mPEGseseCe6PBAP conjugates). Nanophotosensitizers of mPEGseseCe6PBAP conjugates were prepared by dialysis method. In transmission electron microscope (TEM) observation, nanophotosensitizers of mPEGseseCe6PBAP conjugates have spherical shapes and their diameters were less than 150 nm. The average diameter of mPEGseseCe6PBAP nanophotosensitizers was 92.7 ± 9.6 nm in particle size analysis. When H2O2 was added to the nanophotosensitizer solution, nanophotosensitizers were sensitively disintegrated according to the H2O2 concentration and then changed from monomodal distribution to multimodal distribution in particle size distribution. Furthermore, Ce6 release from nanophotosensitizers also increased according to the H2O2 concentration. When H2O2 was added to cell culture of HeLa human cervical cancer cells, intracellular Ce6 uptake of nanophotosensitizers were gradually increased according to the H2O2 concentration, indicating that nanophotosensitizers showed ROS-sensitive delivery of Ce6 against cancer cells.As well as free Ce6, nanophotosensitizers in the absence of light irradiation have low intrinsic cytotoxicity against RAW264.7 cells and HeLa cells. However, nanophotosensitizers induced cell death dose-dependently under light irradiation. Especially, nanophotosensitizers showed significantly higher ROS generation and phototoxicity against HeLa cells in vitro. When nanophotosensitizers were intravenously administered to animal tumor xenograft model of HeLa cells, tumor tissues revealed stronger fluorescence intensity than other tissues by light irradiation while absence of light irradiation induced relatively lower fluorescence intensity in tumor tissues, indicating that nanophotosensitizers have sensitivity against oxidative stress in tumor tissues. We suggest that nanophotosensitizers of mPEGseseCe6PBAP conjugates are promising vehicle for PDT of cervical cancer cells.

Three-Dimensional Human Alveolar Stem Cell Culture Models Reveal Infection Response to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the cause of a present pandemic, infects human lung alveolar type 2 (hAT2) cells. Characterizing pathogenesis is crucial for developing vaccines and therapeutics. However, the lack of models mirroring the cellular physiology and pathology of hAT2 cells limits the study. Here, we develop a feeder-free, long-term, three-dimensional (3D) culture technique for hAT2 cells derived from primary human lung tissue and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and proinflammatory genes in infected hAT2 cells, indicating a robust endogenous innate immune response. Further tracing of viral mutations acquired during transmission identifies full infection of individual cells effectively from a single viral entry. Our study provides deep insights into the pathogenesis of SARS-CoV-2 and the application of defined 3D hAT2 cultures as models for respiratory diseases.

Combinatorial Effect of Cold Atmosphere Plasma (CAP) and the Anticancer Drug Cisplatin on Oral Squamous Cell Cancer Therapy.

Cold atmospheric plasma (CAP) has been extensively investigated in the local treatment of cancer due to its potential of reactive oxygen species (ROS) generation in biological systems. In this study, we examined the synergistic effect of combination of CAP and cisplatin-mediated chemotherapy of oral squamous cell carcinoma (OSCC) in vitro. SCC-15 OSCC cells and human gingival fibroblasts (HGF-1) cells were treated with cisplatin, and then, the cells were irradiated with CAP. Following this, viability and apoptosis behavior of the cells were investigated. The viability of SCC-15 cells was inhibited by cisplatin with a dose-dependent manner and CAP treatment time. HGF-1 cells also showed decreased viability by treatment with cisplatin and CAP. Combination of 1 µM cisplatin plus 3 min of CAP treatment or 3 µM cisplatin plus 1 min of CAP treatment showed a synergistic anticancer effect with appropriate cytotoxicity against normal cells. ROS generation and dead cell staining were also increased by the increase in CAP treatment time. Furthermore, tumor-suppressor proteins and apoptosis-related enzymes also increased according to the treatment time of CAP. We showed the synergistic effect of cisplatin and CAP treatment against SCC-15 cells with low cytotoxicity against normal cells.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Stability in Multiple Water Matrices.

The advent of the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates a thorough study of the stability and transmissibility in the environment. We characterized the stability of SARS-CoV-2 in three water matrices: fresh, tap, and seawater. The minimum infective dose of SARS-CoV-2 in Vero cells was confirmed to be 10³ PFU/mL. The stability of SARS-CoV-2 varied according to the water matrix: infective SARS-CoV-2 was undetectable after treatment with fresh water and seawater, but remained detectable for 2 days in tap water, when starting with an initial concentration of 104 PFU/mL. When the starting concentration was increased to 105 PFU/mL, a similar trend was observed. In addition, viral RNA persisted longer than infectious virus in all water matrices. This study was conducted in stagnant water containing a significantly high titer of virus, thus, human-to-human transmission of SARS-CoV-2 through the actual aquatic environment is expected to be rare.