Angiopep-2、IP10-EGFRvⅢscFv Fusion Protein Modified Nanoparticles Combining With CTL Treat Malignant Glioma

Chapter I Construction and preparation of fusion protein IP10-EGFRvIIIscFvObjective To construct cytokine fusion protein IP10-EGFRvIIIscFv through genetic engineering techniques. To accomplish the large amplification、purification and refolding of fusion protein. Methods To construct the eukaryotic recombinant expression plasmids, GV219IP10-EGFRvIIIscFv。To connect humanIP10and human EGFRvIIIscFv genes with the sequences encoding a flexible linker of (Gly4Ser)3, and subcloned sequentially into downstream of promoter of CMV in expression vector GV219. The target gene IP10-EGFRvIIIscFv in the eukaryotic recombinant expression plasmids was cut by restriction endonuclease and cloned into T7promoter downstream. Then efficiently expressed recombinant protein under IPTG induction. To purify the fusion protein IP10-EGFRvIIIscFv trough utilizingNi-His tag Fusion Protein Purification Kit. To fefold the fusion protein through dialysis. In order to improve the yield of refolded protein, we introduced the oxidation conditions by adding GSSG/GSH to the dialysis buffer. To detect the fusion protein purity through silver staining. To analysis the fusion protein through WB. Results eukaryotic recombinant expression plasmid, GV219IP10-EGFRvIIIscFv was successfully constructed. A large number of fusion protein were obtained(inclusion body expression). The purity of fusion protein was>95%througu MACS, and the MW was46kDa through silver staining and western-blotting. Chapter Ⅱ Preparation of Angiopep-2、IP10-EGFRvⅢ scFv fusion protein modified nanoparticles and brain across testObject To prepare the Angiopep-2、IP10-EGFRvⅢscFv fusion protein modified nanoparticles through nanotechnology. To accomplish in vitro and in vivo tests of brain penetration. Methods Polymer nanoparticles were prepared by utilzing biological nanomaterial poly(lactic-co-glycolic acid(PLGA) and PLGA-PEG-Mal. Meanwhile, we utilized Angiopep-2and/or IP10-scFv modifing the surface of nanoparticles, then we obtain AINPs (as experimental group) and ANPs、INPs、NPs、(as control group). To evaluate the brain drug delivery of AINPs though utilizing coumarin-6and rhodamine-B as fluorescent probe. We injected the fluorescent nanoparticles labled with coumarin-6to tail vein of BABL/C/Nude nu, follow by confocal imaging to evaluate the brain drug delivery of AINPs. We injected the fluorescent nanoparticles labled with DiR to tail vein of BABL/C/Nude nu, follow by vivo imaging to evaluate the brain drug delivery of AINPs.Results The nanoparticles were generally spherical and uniform, nanoparticle sizes were corresponded with the mean particle size determined by DLS. In bEnd.3cells, the fluorescence intensity of INPs was the same as NPs, while that of ANPs and AINPs was higher than that of NPs and INPs, suggesting the angiopep-2could increase the uptake of NP while IP10-EGFRvⅢscFv could not. The fluorescence intensity of NPs and INPs was obviously lower than that of ANPs and AINPs, especially at0.5h and2h post injection, indicating that angiopep-2could facilitate the transport of nanoparticles into brain, but the IP10-EGFRvⅢscFv could not. Through in vivo and in vitro experiments, described AINPs we prepared could effectively across the blood brain barrier. Chapter III Functional studies of Angiopep-2. IP10-EGFRvIII scFv fusion protein modified nanoparticles combining CTL to treat Malignant gliomaObjective To Evaluate AINPs in vitro chemotactic properties and characteristics of targeting glioma cells and glioma experimental treatment combined with CTL. Methods We detected in vitro chemotactic properties of AINPs through Transwell cell migration assays, and detected antigen binding through immunofluorescence staining. Cytotoxic lymphocyte(CTL) was harvested by gliomas cells lysated-pulsed dendritic cells co-cultured with CD8+T lymphocyte. Then combined with AINPs via the tail vein injection BABL/C/Nude nu-situ glioma model. By synergistic action, the change of tumor volume and the survival time of tumor-bearing mice were recorded. Results AINPs effective CD8+T lymphocyte chemotactic, and effectively identificate and bined with the specific antigen EGFRvIII locating on cell lines U87-EGFRvIII. We successfully prepared glioma-specific cytotoxic T lymphocyte(CTL) through DC co-culture techniques. CTL reached the maximum concentration of infiltration around gliomas under the chemotaxis of AINPs, and effectivele killed malignant glioma cells.