Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice.

BACKGROUND & AIMS: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. METHODS: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. RESULTS: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. CONCLUSIONS: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Direct electrochemistry and electrocatalysis of hemoglobin in poly-3-hydroxybutyrate membrane.

Hemoglobin (Hb) can take direct electron-transfer reactions after being entrapped in poly-3-hydroxybutyrate (PHB) film. A pair of well-defined, quasi-reversible cyclic voltammetric peaks is thus obtained at an Hb-PHB modified pyrolytic graphite electrode. The anodic and cathodic peaks are located at -224 and -284 mV for a pH 5.0 acetate buffer solution. Meanwhile, the peroxidase activity of the protein in the membrane has been greatly enhanced, with the apparent Michaelis-Menten constant calculated to be 1076 microM. According to the direct electron transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb-PHB based hydrogen peroxide biosensor is prepared, with a linear range 6.0 x 10(-7) to 8.0 x 10(-4) M. The pathway of reductive dehalogenation of trichloroacetic acid is also discussed in detail. The highly reduced form of Hb produced in PHB film can be used to dechlorinate di- and monochloroacetic acid. The catalytic ability of Hb toward the reduction of nitric oxide has been investigated as well. Due to its biodegradability, low cost, chemical inertness, and especially its biocompatibility and non-toxicity, PHB would be a desirable film in the sensor field.

A new film for the fabrication of an unmediated H2O2 biosensor.

A novel and stable film made from polyethylene glycol (PEG) on pyrolytic graphite (PG) electrode was presented in this paper for incorporating horseradish peroxidase (HRP) to study the direct electrochemistry of the enzyme. In PEG film, HRP showed a thin-layer electrochemistry behavior. The apparent standard potential (E degrees ') was -0.379 V versus SCE at pH 7.2. Moreover, the PEG-HRP modified electrode exhibited excellent electrocatalytical response to the reduction of H2O2 with a calibration range between 2.0 x 10(-6) and 6.0 x 10(-4) M and a good linear relation from 2.0 x 10(-6) to 1.0 x 10(-4) M, on which an unmediated H2O2 biosensor was based. The detection limit of 6.7 x 10(-7) M was estimated when the signal-to-noise ratio was 3. The relative standard deviation (R.S.D.) was 4.7% for six successive determinations at a concentration of 4.0 x 10(-5) M. The apparent Michaelis-Menten constant (Km app) of the sensor was found to be 1.38 mM. Epinephrine, dopamine, and ascorbic acid did not interfere with the sensitive determination of H2O2.

A nitric oxide biosensor based on the multi-assembly of hemoglobin/montmorillonite/polyvinyl alcohol at a pyrolytic graphite electrode.

Direct electron transfer of hemoglobin (Hb) can be achieved in a Hb/montmorillonite (MMT)/polyvinyl alcohol multi-assembly at a pyrolytic graphite (PG) electrode. Accordingly, a novel nitric oxide (NO) biosensor is proposed. The reduction of NO is observed at a potential of approximately -783 mV (vs. SCE) at pH 5.5. At optimum pH, this biosensor shows a wide linear range of 1.0x10(-6)-2.5x10(-4) mol/l with a detection limit of 5.0x10(-7) mol/l. The sensor-to-sensor reproducibility is good consideringmacr; a relative standard deviation of 3.5% in five independent determinations at 5.0x10(-5) mol/l NO. The modified electrode is conveniently constructed and durable in long-term operations.

Enhanced electron-transfer reactivity of horseradish peroxidase in phosphatidylcholine films and its catalysis to nitric oxide.

Direct electrochemistry of horseradish peroxidase (HRP) embedded in film of phosphatidylcholine (PC) is investigated at a pyrolytic graphite electrode by voltammetric methods. The electron-transfer reactivity between incorporated HRP and the electrode is found to be greatly enhanced by phosphatidylcholine film. Cyclic voltammetry (CV) of this incorporated peroxidase shows a pair of well-defined and nearly reversible peaks, and the cathodic and anodic peak potentials are located at about -0.261 and -0.180 V, respectively versus saturated calomel electrode at pH 5.5. Ultraviolet-visible absorption spectra indicate that the heme microenvironment of HRP in phosphatidylcholine film is similar to that of its native status. It is also observed that HRP modified electrode is able to catalyze the electrochemical reduction of nitric oxide. Experimental results reveal that the peak current related to nitric oxide reduction is linearly proportional to its concentration in the ranges of 2.0 x 10(-7) -5.0 x 10(-6) mol (-1) and 2.0 x 10(-5) -1.0 x 10(-4) mol(-1), based on which an unmediated biosensor for nitric oxide is developed.

A reagentless nitric oxide biosensor based on haemoglobin/polyethyleneimine film.

An unmediated NO biosensor was prepared by co-immobilizing haemoglobin with polyethyleneimine at a pyrolytic graphite electrode surface. Haemoglobin exhibits direct electrochemistry in the membrane, and this modified electrode shows high stability and catalytic activity towards the reduction of NO. A linear relationship between the catalytic current and the concentration of NO was observed in the concentration ranges 1.0 x 10(-7)-8.0 x 10(-6) M and 1.0 x 10(-5)-2.5 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M.