R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells.

The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ±â€¯42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14+ macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.

Preparation of the phycoerythrin subunit liposome in a photodynamic experiment on liver cancer cells.

AIM: Efforts are underway to establish a preparation method for the phycoerythrin subunit (PE-sub) liposome, and enhance the cellular uptake and photodynamic therapy (PDT) effect on cancer cells. METHODS: A film dispersion method was used to prepare the PE-sub liposome, an orthogonal analysis was conducted to optimize the PE-sub liposome preparation condition and determine the effects of liposomes as carriers on cell uptake in vitro. Under a fluorescence microscope, the cell survival rate of normal liver cell line HL7702 and liver cancer cell line HepG2 was assessed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. Cell apoptosis was determined with flow cytometry and acridine orange staining after PDT treatment. RESULTS: The optimum preparation conditions of the PE-sub liposome were found: a phosphatidylcholine-to-cholesterin ratio of 1:2, a PE-sub-to-lipid ratio of 1:30, 20 mL buffer volume, 10 min sonication time, and an average encapsulation rate of up to 47.2%. The particle size ranged from 80 to 200 nm, and the average particle diameter was 136 nm. At a concentration of 100 microg/mL, the transfection rate of the PE-sub liposome reached 18% at 2 h and 24% at 4 h, and remained steady at 5-6 h. The half lethal dose of PDT on HepG2 was 75 microg/mL, whereas the cell survival rate of HL7702 reached 80% at the same dosage. The PDT-treated cells showed characteristics of apoptosis. CONCLUSION: The film dispersion method was found to maintain the biological characteristics of the PE-sub. The use of the liposome carrier increased the PE-sub accumulation in the cells and enhanced its PDT effect on HepG2 compared to the PE-sub. HL7702 cell toxicity on had less apparent change after PDT treatment. The PE-sub liposome demonstrated good tumor-targeting characteristics in the in vitro experiment.

The experimental research of R-phycoerythrin subunits on cancer treatment: a new photosensitizer in PDT.

The mouse tumor cell S180 and human liver carcinoma cell SMC 7721 cells were first treated with R-PE and its subunits (alpha, beta, gamma subunits), then irradiated with Argon laser (496 nm, 28.8 J/cm2). Survival rate was measured by MTT method. In order to compare the phototoxicity in normal cells, the mouse marrow cells were treated with photofrin II and beta-subunit, irradiated with 45 J/cm2 of light; survival rate was also measured by MTT method. The result showed that R-PE subunits had better PDT effect on s180 cells than R-PE and lower phototoxicity in marrow cells than photofrin II. Flow cytometric analysis showed that PDT results in a growth inhibition and a G0-G1 cell cycle arrest in SMC 7721 cells. The tumor cells inhibited by PDT in vivo were morphologically observed by TEM, the tumor cell death was due to the occlusion of tumor blood vessels and inducement of cell programmed death in nuclei. Therefore, with the advantage in special fluorescence activity, low molecular weight, good light absorbent character and weak phototoxicity, R-PE subunit is an attractive option for improving the selectivity of PDT.