The Influence Of Nanoemusion/nanoliposome On DC

A great deal of drug nano-carriers have been produced by use of nanotechnology. Nano-carriers have many excellent characters and new functions because of the particular properties of nano-particles. Nowadays, many kinds of drug nano-carriers have been used to design and produce tumor vaccines successively.In order to make nano-vaccine work effectively, drug nano-carriers must be intaken and presented by antigen presenting cells (APC) as first steps. Although the antigen proteins, peptides, antigen-encoding genes are the effective components of nano-vaccines, drug nano-carriers are also intaken by APC. During this process, the activity and function of APC may be affected because of drug nano-carriers itself. The application of nanoemusion and nanoliposomes as drug nano-carriers is to have prosperous future. Workmates in our lab have prepared tumor specific antigen enwrapped by nanoemusion and nanoliposomes respectively, both of which manifest sound effect in animal experiment. By the following assays in vivo, we found that over-loaded nanoemusion as a carrier is toxic to mice. As the nanoemusion beyond a certain level is injected to mice, the death rate of treatment group is high than the death rate of contral group. Dendritic cells (DC) are the most potent professional antigen presenting cells and the exclusive cells which can activate the resting T cells. The drug delivery system of nano-vaccine tagetting DC is to be a new study direction in the nanomedicine field. The reseach about toxicity and activity of drug nano-carriers to human DC has not been reported by far. We established the methods to culture DC from human peripheral blood mononuclear cells and peripheral blood stem cells in vitro. Then we discuss the influence of nanoemusion and nanoliposomes to DC from PBMC. By these experiments we lay a solid foundation for the in-vivo reseach of nano-vaccine targeting DC.1 Culturing dendritic cells from human peripheral blood mononuclear cells and human peripheral blood stem cells.Objective: To improve the method of isolated cultivation of dendritic cells from the peripheral blood mononuclear cells, and get comparatively large amount of DC with high purity. To establish the method of culturing dendritic cells from the peripheral blood stem cells which is released into peripheral blood by G-CSF mobilization. Methods: Utilizing successive adherence method to obtain CD14+ precursor cells. The mature DC was induced in vitro by granuloeyte-macrophage colony-stimulating factor(GM-CSF), interleukin-4(IL-4) and tumor necrosis factor -α(TNF–α). The marrow cells of healthy donors are mobilized by granuloeyte colony-stimulating factor (G-CSF). After four days, harvesting peripheral blood stem cell with Cobe SpectraTM blood collection machine as the precursory cells of dendritic cells. Determining the expression level of several DC surface markers by flow cytometry. Observing the morphological features and subcellular structure of dendritic cells from PBMC with transmission electron microscope (TEM) and scan electron microscope(SEM). Results: The quantity and purity of DC isolated from human PBMC with successive adherence method is more satisfactory than that of DC isolated with traditional one-time adherence method .We obtained 0.5～1×10~7 DC from 10~8 PBMC with successive adherence method, and the purity of obtained DC is over 80%. The DC cultured 1 week highly expresses CD1a(44.16%), CD40(75.04%), HLA-DR(95.98 %), CD83(57.25) and CD86(73.20%). It is observed by SEM that the shape of DC is irregular: circular, oval, diamond or polygonal etc. The diameter of DC body is over 10μm. Tiny villus is enriched in the surface. Numerous dendrites protrude radially from cell bodies, some of which are as long as 2 or 3 times of cell bodies, and the dendrites cross and fuse each other. Many protrusions adhere to the flask bottom and cell bodies suspend. We observed the subcellular structure of dendritic cells in different phases with TEM: the shape of nuclei is irregular : kidney-shaped, oval, U-shaped. Most nuclei locate unsymmetrically. Euchromatin is predominant and heterochromatin can hardly be seen. Nuclear membranes are clear and nucleoli can easily be found. There are abundant lysosomes and developed Golgi complex in the cytoplasm of DC from PBMC and affluent chondrosomes surrounding RER. Apoptosis and phagotrophy can be detected obviously on the 15th day in cultured DC. More DC can be obtained from PBSC after G-CSF administration. At least 10~7 DC can be harvested from 5ml separating medium containing large amount of PBSC. Longer period ( 9 days ) and more cytokines are needed in the culturing of DC from PBSC compared with those from PBMC.The phenotype and morphological features of DC from PBSC are similar with those from PBMC and certain surface markers are also highly expressed in DC from PBSC, of which CD1a(48.73%), CD40(85.04%), HLA-DR(91.12 %), CD83(55.11%) and CD86(80.10%). Conclusion: We set up successive adherence method to isolate and culture DC from PBMC by which we get more DC with high purity.Meanwhile, we set up the method to isolate and culture DC from PBSC which is harvested by Cobe SpectraTM blood collection machine after G-CSF administration. By this method we get larger amount of DC and waste fewer components of blood other than DC. After all we consider that the application of DC from PBSC has prosperous future.2.The influence of nanoemusion /nanoliposome on DCObjective: To determine the toxicity and activity of nanoemusion /nanoliposome on DC from human PBMC. Method: After culturing of 5 days, immature DC from human PBMC was loaded with different dose of nanoemusion /nanoliposome and then incubated for another 48 hours. The morphological changes of DC were observed by optics microscope. Detecting the expression level of DC surface marker by flow cytometry. Observing the morphological and subcellular structur changes of dendritic cells loaded with nanoemusion/nanoliposome by TEM and SEM. MTT assay was applied to determine the capability of DC loaded with nanoemusion/nanoliposome stimulating proliferation of T lymphocyte respectively. Results:①Nanoemusion group: After 100μl nanoemusion was added into 1×10~5/ml DC, severe swelling, disaggregation and necrosis of DC was detected by optics microscope progressively. After 20μl, 30μl or 50μl nanoemusion was added into 1×10 5/ml DC, cellular swelling was detected by optics microscope and cell organelles engorgement and nuclei enlargement took place. There are many lipid-droplet in the swelling cells. Cell menbrane of little amount of DC collapsed and disaggregation and necrosis of DC were detected seldomly. After 10μl nanoemusion was added into 1×10 5/ml DC, cell shape remained still and phagolysosomes enwrapping nanoemusion was found by TEM. The expression level of DC surface marker CD83 was up-regulated in the DC loaded with 10μl nanoemusion. The result of MTT assay showed that the capability of DC stimulating proliferation of T lymphocyte descended obviously.②Nanoliposome groups: After 50-100μl nanoliposome was added into 1×10 5/ml DC, the shape of DC became circular and the amount of surface protuberances on DC membrane decreased. Lipidoses and intracellular cholesterol crystal can be found in endochylema of DC notably by TEM. But necrosis and apoptosis were not found in DC of this group. The expression level of DC surface marker CD83 remained still by FCM. The MTT assay manifests that the capability of DC stimulating proliferation of T lymphocyte is not different from the contral group. Conclusions: nanoemusion with proper dose can be used as a drug carrier efficiently intaken by DC and can promote DC maturation and differentiation. But cellular swelling and necrosis appears as nanoemusion with volume more than 20μl is added into 1×105 /ml DC, showing that excessive nanoemusion is toxic to DC. The morphological feature and activity of DC loaded with 100μl nanoliposome is not affected obviously, suggesting that naoliposome is more tolerable to DC than nanoemusion.In the first part of experiment, we established the successive adherence method to culture DC from PBMC and cultured DC from PBSC which is harvested by blood collection machine following G-CSF administration. We found that DC from PBMC and PBSC have typical molecular phenotype and morphological feature, normal ultrastructure and function. In the second part of experiment, we testified that toxicity and influence on DC activity of nanoemusion are more severe than to DC than that of nanoliposome. It suggests that the mechanism of DC swelling may be associated with surface acting agent in nanoemusion (further investigation is needed to testify this conclusion). Nanoliposome is almost non-toxic to DC and has no effect on DC activity differing from nanoemusion. With these findings, we can conduct further investigation on tumor vaccine based on nanoliposome targeting DC.