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The Study Of Biotin-conjugated Pullulan Acetate Nanoparticles As A Novel Anticancer Drug Carrier

Posted on:2019-03-22Degree:MasterType:Thesis
Country:ChinaCandidate:X J WeiFull Text:PDF
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BackgroundWith the development of nanotechnology,nanomaterial-based products have shown promise in cancer treatments and have been approved for the clinical research.Due to its excellent bioproperties,pullulan has been recently used as the carrier material to prepare the targeted drug delivery systems.In our previous studies,pullulan acetate nanoparticles that we previously prepared had excellent drug loading and sustained drug release properties.Biotin receptor is expressed on malignant cells at levels several fold higher than those on normal cells and the necessity for cancer cell proliferation.Thus,biotin has been generally used as an effective cancer-targeting ligand to modify the polymeric prodrugs and the nanodrug carriers.Herein,we synthesized biotin grafted pullulan acetate(Bio-PA)copolymer,which focused on obtaining a tumor targeted drug delivery carrier.Objective1.In this study we explored the feasibility of the novel prepared Bio-PA NPs as drug carriers.2.We also clarified the role of Bio-PA NPs targeting anti-tumor effect and studied the mechanism of inhibition of tumor.3.Established a xenograft model by implanting HepG2 3D cultured cells system subcutaneously into nude mice and further evaluated the in vivo antitumor activity of BioPA NPs.Methods1.Preparation and characterization of the NPs.Synthesis and Characterization of BioPA and PA;The NPs were synthesied by dialysis and emulsion solvent evaporation method;Particle size and surface potential were measured by particle size analyzer;The morphology of NPs was observed by TEM.The entrapment efficiency,drug loading,in vitro release and the DS of biotin were measured.2.Cytotoxicity assay.The cytotoxicities of Bio-PA NPs in three cells(biotin receptor over-expressed MCF-7 cell,HepG2 cell and the biotin receptor low-expressed 293 T cell)were evaluated using the CCK-8 assay.The uptakes and distributions of the NPs in cells were evaluated by the confocal microscope.And the celluar uptakes were further quantificationally determined by the flow cytometry.3.To investigate the cell entry mechanism of Bio-PA/EPI NPs,their uptakes in HepG2,MCF-7 and 293 T cells,were further detected in the presence of different uptake inhibitors,including CPZ(the clathrin-mediated endocytosis inhibitor),CQ(the endosome-lysosome pathway inhibitor),AMR(a specific Na+/H+ exchange inhibitor),NYS(the caveolae-dependent endocytic pathway inhibitor),FLP(the caveolae-mediated uptake inhibitor)and free biotin using the flow cytometry and fluorescence spectrophotometry methods.4.To explore the cell morphology of three-dimensional(3D)culture modein vitro and two-dimensional(2D)culture by the confocal microscope.We established KM mice tumor model by two strategies,2D cultured HepG2 cells and 3D cultured cells system method.The tumor volume,KM mice weight and histology of the subcutaneous tumor tissue were evaluated.5.In vivo tumor tissue distribution and anti-tumor efficacy.The NPs were labeled with Cy7,then assessed their tumor tissue distributionand after intravenous injections using in vivo photoacoustic Imaging System.The tumor volume changes were determined to evaluate the antitumor effect in vivo.Results1.The 1H NMR results of biotin,pullulan,PA and Bio-PA confirmed biotin has been grafted onto PA backbone successfully.The TEM images showed the NPs had the regularly spherical shapes by the dialysis and solvent diffusion method.The EE,LC and content of biotin on the surface of Bio-PA NPs prepared by dialysis was higher than that of by solvent diffusion method.A sustained drug release pattern was observed,and the amount of EPI released in acidic media(pH 5.5)was higher than in media at physiological pH(pH 7.4).EPI exhibited a rapid initial drug release during the first 12 h,and then followed by a significant sustained release from the Bio-PA NPs.2.In vitro cytotoxic experiments show,two kinds of blank NPs with concentration ranging from 5 ?g/mL to 1000 ?g/mL were co-incubated with three kinds of cells for 24 h and 48 h,and had no significant effect on cell proliferation.Bio-PA/EPI NPs and PA/EPINPs were found to have significant cytotoxicity on cells after drug-loaded nanoparticles and free drug EPI were incubated with three kinds of cells at 0.025,0.25,2.5,10 and 25?g/mL for 24 h and 48 h respectively.Bio-PA/EPI NPs had the best effect,which indicated that it had a good anti-tumor effect in vitro.Cellular uptake studies showed that the clathrin-mediated endocytosis pathway was the main pathway for cellular uptake of nanoparticles.The addition of free biotin reduced the uptake of NPs by 62.4% and 60.5%in MCF-7 and HepG2 cells,whereas 293 T The cells did not change significantly.Confocal images and flow cytometry results show that NPs can be well taken by these cells.3.The results of microscopy and laser confocal microscopy show that HepG2 cells grow in a spherical shape in a 3D hydrogel and can form cell clumps,increasing the interrelationship between cells.The tumor formation experiments in animals showed that the tumor growth rate in the 3D model was about 2 times that of the 2D model,and the weight change was relatively stable and there was no animal death.4.Tumor tissue distribution of the Cy7 labeled the Bio-PA NPs and PA NPs were assessed after intravenous injections using the free Cy7 as a control.At 12 h after injection,free Cy7 could not be detected,indicating that Cy7 was rapidly eliminated,whereas Cy7 labeled Bio-PA NPs and PA NPs were distributed the tumor tissue at same time.The fluorescence intensity in Bio-PA NPs and PA NPs group can continue to the 24 h,and Cy7-Bio-PA is significantly stronger than Cy7-PA,indicating that the Bio-PA NPs have the targeting and long cycle.In vivo antitumor experiments showed Bio-PA/EPI NPs significantly inhibited tumor growth,and the average tumor size was significantly smaller than the control group,PA/EPI NPs treatment group and EPI group.Conclusion1.Bio-PA was successfully synthesized and Bio-PA NPs were prepared.Nanoparticles were uniform in size,round in shape,with high entrapment efficiency and drug loading.EPI release in vitro has sustained release properties.2.There was no significant cytotoxicity in the free-loaded nanoparticles within a certain concentration range.Compared with PA/EPI NPs and EPI,Bio-PA/EPI NPs had higher biotin-expressing cellular uptake and significantly enhanced anti-tumor effect in vitro.3.The HepG2 cells grew in a 3D manner within the hydrogel,which lead to forminghomogenous multicellular spheroids.The 3D hydrogels culture model has the potential to improve cell culture strategies and be used for establishment the subcutaneous tumor model in nude mice.In HepG2 tumor-bearing nude mice,Bio-PA/EPI NPs could the increased distributions and prolonged the circulationtime in the tumor.4.In summary,Bio-PA NPs exhibit good sustained release properties,long circulation,and tumor targeting effect.So,the data presented in this study can therefore be of great value in the development of targeted anticancer drug delivery.
Keywords/Search Tags:pullulan, biotin, nanoparticle, targeted anti-tumor, three-dimensional hydrogels
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