Cisplatin-incorporated nanoparticles of methoxy poly(ethylene glycol)-b-poly(L-glutamic acid) copolymer.

We fabricated cisplatin-incorporated nanoparticles using block copolymer composed of methoxy poly(ethylene glycol) (MPEG) and poly(L-glutamic acid) (PGA) (abbreviated as GE). For synthesis of block copolymer, MPEG was directly conjugated to the terminal amine of PGA. Cisplatin-incorporated nanoparticles was prepared by ion complex formation of cisplatin and PGA domain of block copolymer. Size of cisplatin-incorporated nanoparticles was less than 200 nm at particle size measurement and their shapes were spherical at TEM observation. To study drug release properties, cispaltin release from nanoparticles were performed at phosphate-buffered saline (PBS, 0.01 M, pH 7.4) and distilled water, indicating that cisplatin release rate at PBS was approximately three times higher than that of deionized water. The cell growth inhibition of cisplatin incorporated nanoparticles in vitro was tested with HuCC-T1 cells. Cisplatin-incorporated nanoparticles were effectively inhibited tumor cell growth as similar as cisplatin itself. In other words, nanoparticles showed decreased inherent cytotoxicity compared to cisplatin against normal cells. We suggest that cisplatin incorporated GE nanoparticles is a good candidate for antitumor drug targeting.

Prevention of covered enteral stent migration in patients with malignant gastric outlet obstruction: a pilot study of anchoring with endoscopic clips.

OBJECTIVE: Placement of a self-expandable metal stent is a palliative treatment of choice for patients with malignant gastric outlet obstruction (GOO). Although covering an enteral stent with a membrane almost solves the problem of tumor ingrowth, stent migration continues to be a major unresolved problem. Our aim was to evaluate the clinical efficacy of endoscopic clipping for prevention of covered stent migration in the treatment of malignant GOO. MATERIAL AND METHODS: A total of 25 consecutive patients with malignant GOO were evaluated prospectively. After deployment of a double-layered combination stent (comprising an outer uncovered stent and an inner covered stent), three endoscopic clips were applied for fixation of the proximal end of the enteral stent to the gastric or duodenal mucosa. RESULTS: Technical and clinical success rates were 100% (25/25) and 88% (22/25), respectively. No stent migration was observed in any of the patients. Five patients (20%) experienced complications such as tumor overgrowth and stent compression. CONCLUSION: Endoscopic clipping for enteral stent placement seems to be effective for prevention of covered stent migration in patients with malignant GOO.

Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells.

The mechanisms that underlie the altered neurotransmitter system in Alzheimer's disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for beta-amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11alpha, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP-X11alpha interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion.

5-aminolevulinic acid-incorporated poly(vinyl alcohol) nanofiber-coated metal stent for application in photodynamic therapy.

BACKGROUND: The study investigated the use of combined photodynamic therapy (PDT) and stent placement for the treatment of cholangiocarcinoma (CC). For this purpose, 5-aminolevulinic acid (ALA) was incorporated into poly(vinyl alcohol) (PVA) nanofiber, and coated onto metal stents. Their efficacy was assessed in PDT towards HuCC-T1 CC cells. METHODS: Fabrication of ALA-PVA nanofiber, and simultaneous coating onto metal stents, was performed through electrospinning. The dark-toxicity, generation of protoporphyrin IX (PpIX), and PDT effect of ALA and ALA-PVA nanofiber were studied in vitro, using HuCC-T1 CC cells. RESULTS: The ALA-PVA nanofibers were coated onto metal stents less than 1000 nm in diameter. ALA-only displayed marginal cytotoxicity; ALA-PVA nanofiber showed less cytotoxicity. PpIX generation was not sigficantly different between ALA and ALA-PVA nanofiber treatments. PVA itself did not generate PpIX in tumor cells. ALA and ALA-PVA nanofiber displayed a similar PDT effect on tumor cells. Cell viability was decreased, dose-dependently, until ALA concentration reached 100 µg/mL. Necrosis and apoptosis of tumor cells occurred similarly for ALA and ALA- PVA nanofiber treatments. CONCLUSION: The ALA-PVA nanofiber-coated stent is a promising candidate for therapeutic use with cholangiocarcinoma.

Self-assembled nanoparticles of hyaluronic acid/poly(DL-lactide-co-glycolide) block copolymer.

We synthesized block copolymer composed of hyaluronic acid (HA) and poly(DL-lactide-co-glycolide) (PLGA) (HAbLG) for antitumor targeting. (1)H NMR was employed to confirm synthesis of block copolymer. At (1)H NMR study, HabLG nanoparticles showed HA intrinsic peaks only at D(2)O, indicating that they contained HA as a hydrophilic outer-shell and PLGA as a inner-core. Anti-tumor activity was studied using CD44-overexpressing HCT-116 human colon carcinoma cells. Addition of doxorubicin (DOX)-incorporated nanoparticles to tumor cells resulted in the expression of a strong red fluorescence color while they expressed very weak fluorescence when CD44 receptor was blocked with free HA. Flow cytometry data also showed similar results, indicating that the fluorescence intensity of tumor cells treated with nanoparticles was significantly decreased when CD44 receptor was blocked. These results indicate that HAbLG nanoparticles were able to target CD44-overexpressing tumor cells via receptor-mediated endocytosis.

Effect of surfactant on 5-aminolevulinic acid uptake and PpIX generation in human cholangiocarcinoma cell.

Photodynamic therapy (PDT) is a palliative therapy and has been used to cure cholangiocarcinoma (CC), which has a poor prognosis and limited available curative therapy. PDT was shown to improve the median survival time of advanced-stage patients. Recently, 5-aminolevulinic acid (ALA) has been used as a pro-photosensitizer, which can be transferred to intercellular protoporphyrin IX (PpIX), which is a strong photosensitizer, via the heme pathway. The main limitation of using ALA in PDT is the hydrophilic properties of ALA, which results in low cellular uptake. In this study, non-ionic surfactants, pluronic F68 (PF68) and Tween 80 (TW80), were used to address this limitation. The human CC cell line, HuCC-T1, was cotreated with ALA and different concentrations of surfactants for 4h. The effect of surfactants was evaluated by monitoring the uptake of ALA, the fluorescence intensity of PpIX, and the cell survival rate after suitable light irradiation. Cotreatment with the surfactant resulted in an increased intracellular ALA level, PpIX formation, and phototoxicity.

Effect of 5-aminolevulinic acid-based photodynamic therapy via reactive oxygen species in human cholangiocarcinoma cells.

Cancer cells have been reported to exhibit an enhanced capacity for protoporphyrin IX (PpIX) synthesis facilitated by the administration of 5-aminolevulinic acid (ALA). We investigated the effect of ALA-based photodynamic therapy (PDT) on human cholangiocarcinoma cells (HuCC-T1). Since protoporphyrin IX (PpIX), a metabolite of ALA, can produce reactive oxygen species (ROS) under irradiation and then induce phototoxicity, ALA-based PDT is a promising candidate for the treatment of cholangiocarcinoma. When various concentrations of ALA (0.05-2 mM) were used to treat HuCC-T1 cells for 6 or 24 hours, the intracellular PpIX level increased according to the ALA concentration and treatment time. Furthermore, an increased amount of PpIX in HuCC-T1 cells induced increased production of ROS by irradiation, resulting in increased phototoxicity.

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer.

BACKGROUND: Polymeric micelles using amphiphilic macromolecules are promising vehicles for antitumor targeting. In this study, we prepared anticancer agent-incorporated polymeric micelles using novel block copolymer. METHODS: We synthesized a block copolymer composed of dextran and poly (DL-lactide-co-glycolide) (DexbLG) for antitumor drug delivery. Doxorubicin was selected as the anticancer drug, and was incorporated into polymeric micelles by dialysis. Polymeric micelles were observed by transmission electron microscopy to be spherical and smaller than 100 nm, with a narrow size distribution. The particle size of doxorubicin-incorporated polymeric micelles increased with increasing drug content. Higher initial drug feeding also increased the drug content. RESULTS: During the drug-release study, an initial burst release of doxorubicin was observed for 10 hours, and doxorubicin was continuously released over 4 days. To investigate the in vitro anticancer effects of the polymeric micelles, doxorubicin-resistant HuCC-T1 cells were treated with a very high concentration of doxorubicin. In an antiproliferation study, the polymeric micelles showed higher cytotoxicity to doxorubicin-resistant HuCC-T1 cells than free doxorubicin, indicating that the polymeric micelles were effectively engulfed by tumor cells, while free doxorubicin hardly penetrated the tumor cell membrane. On confocal laser scanning microscopy, free doxorubicin expressed very weak fluorescence intensity, while the polymeric micelles expressed strong red fluorescence. Furthermore, in flow cytometric analysis, fluorescence intensity of polymeric micelles was almost twice as high than with free doxorubicin. CONCLUSION: DexbLG polymeric micelles incorporating doxorubicin are promising vehicles for antitumor drug targeting.

DYRK1A-mediated hyperphosphorylation of Tau. A functional link between Down syndrome and Alzheimer disease.

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD.