| Background:Tumour immunotherapy has been a very promsing treatment nowadays since more and more studies have focused on the immune pathogenesis of cancer.Compared with radiotherapy and chemotherapy,tumour immunotherapy has a potent anti-tumour effect without severe side effects.As an important immune component,the antigen presenting cells play an important role in the tumour progression.The antigen presenting cells are a group of cells which are able to uptake,digest and present antigens.Traditional antigen presenting cells are composed of B cells,macrophages and dendritic cells(DCs).The important role of these cells in tumour immunology has d RAW 264.7n much attention.DCs are a kind of professional antigen presenting cells(APCs).Immature DCs have a strong ability to uptake antigen while mature DCs show a more potent ability to present antigens.The dysfunction of DCs can be generally observed in tumour-bearing status including a block of maturation and a lower expression of MHC II and some co-stimulate molecules.All of the dysfunctions lead to a poor antigen presenting which prevents the activation of T cells.The macrophages can both present antigens and secret different cytokines to regulate the immune response which play an important role in the crosstalk between innate immunity and adaptive immunity.In tumour-bearing status,the macrophages transmute a polarization towards M2 phenotype and become the tumour associated macrophages(TAM).These transformed macrophages lose the main function of presenting antigens.Instead,they can secret the cytokines such as IL-10,IL-6 to suppress the anti-tumour effectors and promote the angiopoiesis of tumour.The dysfunction of macrophages and DCs is an important process of the tumour progression.Fortunately,the changes of these cells are reversible.They can be re-educated into normal cells to regain the anti-tumour function by some cell biological method.So it provides us an important chance to transform the antigen presenting cells for tumour treatment.Many remedies have been reported,such as cocultured the DCs isolated from bone marrow with tumour cells,and then transfused the DCs into the body bearing the same tumour.Some reports also showed that blocking the STAT3 and JAK2 pathway with WP1066 could transform the tumour associated macrophages into normal macrophages.These therapies targeting DCs and macrophages have achieved a prominent anti-tumour potency.However,some of the therapies need the cells from vivo and transform in vitro and many of them just have a single target.It is a barrier in clinical practice and limit the treating effect.Cationic polymer(such as protamine,PEI,chitosan,DEAE-dextran)has been widely used in the DNA delivery system in vivo.The molecule has a positive potential.It can aggregate with plasmid DNA and form into nanoparticles stably.The cationic polymer has an “endosome escape” function.It can protect the DNA from the digestion of endosome in the cytoplasm.Undoubtedly,it is a useful tool in the transformation of the antigen presenting cells in vivo.Objective:To construct a tumour therapy which can achieve the potent transformation of antigen presenting cells in vivo by a simple injection of cationic polymer particles.Methods:At first we used Au nanoparticles with different sizes(5 nm,30 nm,50 nm)as a model to research the distribution of nanoparticles in the body(normal mice or 4T1 tumour-bearing ones)with flow cytometry and Caliper IVIS Lumina II.It would be a reference for the following experiments.Then we got a p CMV-EGFP plasmid and constructed a p CMV-c-myc plasmid.So the expression of EGFP and c-myc would be the indicator of the transformation effect.We used the DNA-protamine-liposome nanoparticles and DNA-PEI-PLGA microparticles as the delivery of the plasmid and transfected the RAW 264.7 cell line and peritoneal macrophages in vitro.48 hours and 72 hours later,we analysed the expression of EGFP and c-myc with a fluorescence microscope and the flow cytometry.To evaluate the transformation effect in vivo,we infused the particles carrying p CMV-EGFP or p CMV-c-myc plasmid into normal C57BL/6 mice or ones bearing EG7 lymphoma by intravenous or intraperitoneal injection.48 hours or 72 hours later,the mice were sacrified and the immune organs and tumours were collected.The expression of EGFP or c-myc in the antigen presenting cells was analysed by flow cytometry.Results:By the analyzation of the Au nanoparticles distribution in the body,we found that the immune environments between normal mice and tumour bearing ones were quite different.In tumour bearing mice,the percentage MDSCs in the blood and spleen increased compared with normal ones and the proportion of these cells increased with the augment of tumour size.While the percentage of CD8 postive T cells,B cells and NK cells decreased.The increase of other myloid cells in the blood and spleen could also be observed.The distributions of Au nanoparticles in normal mice and tumour bearing ones were also different.After the implantation of 4T1 tumour,decreased distribution in each population of monocytes in spleen and marrow was observed together with the decreased distribution in the G-MDSCs in the blood.With the augment of the tumour size,the nanoparticles in all sizes accumulated more in the CD8+ T cells,macrophages,DCs and MDSCs in the tumour.And the 5 nm nanoparticles also have a significant accumulation in the NK cells.In the tumour cells,5 nm and 50 nm nanoparticles accumulated more in big tumour compared with the opposite distribution of 30 nm nanoparticles.Referring the result of Au nanoparticle distribution,we began to study the transformation effect of DNA-protamine-liposome.In vitro,we transfected the RAW 264.7 cell lines with DOTAP-cholesterol liposome carring p CMV-EGFP plasmid.72 hours later,by flow cytometry we found that the RAW 264.7 cells could express EGFP with a low quantity.In vivo,we infused the same nanoparticles into normal C57BL/6 mice.48 hours later the MFI of EGFP in the myloid cells of blood increased slightly compared with glucose control.72 hours later,the mice were sacrified and cells from the immune organs were analyzed by flow cytometry.No obvious expression was observed in the myloid cells.Then we changed the composition of the liposome into lecithin and cholesterol and transfected the RAW 264.7 cells and peritoneal macrophages with the new liposome nanoparticles carrying p CMV-c-myc plasmid.48 hours later,we observed the expression of c-myc on the cell surface with a low quantity.But in vivo,we found no expression of c-myc on the myloid cells in the normal or tumour-bearing mice by intravenous or intraperitoneal injectionTo improve the transfection effect,we synthesized a DNA-PEI-PLGA microparticles as the delivery of p CMV-c-myc plasmid.The transfection effect of peritoneal macrophages with the microparticles in vitro increased significantly.The positive percentage of c-myc could increase up to 10.5%.In vivo,48 hours after the peritoneal injection of microparticles with a larger content of PEI,a significant higher positive percertage of c-myc on the peritoneal myloid cells including MDSCs could be observed in the normal mice.In the EG7 tumour-bearing ones,we also achieved the high transfection effect in the peritoneal myloid cells by continuous peritoneal injection for 4 days with the microparticles carrying p CMV-c-myc plasmid with a larger content of PEI.Conclusion:1.The immune environments in the normal mice and tumour-bearing ones were different,which leaded to the different distribution of Au nanoparticles in the body.The distribution of Au nanoparticles was not size dependent.2.RAW 264.7 cells and peritoneal macrophages could express EGFP or c-myc with a low quantity after transfection with DNA-protamine-liposome nanoparticles carrying p CMV-EGFP or p CMV-c-myc plasmid.And the transformation effect in the normal mice and EG7 tumour bearing ones was still low.3.The transfection effect of DNA-PEI-PLGA in vitro and in vivo increased significantly.A high transformation effect could be achieved in tumour-bearing mice by continuous peritoneal injection. |
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