The Study Of Double-layer Core-shell Structure Nanoparticle Combination Chemotherapy For Breast Neoplasms

Background Tumor angiogenesis is critical for tumor growth,invasion and metastasis such as breast cancer.According to previous studies by our group,double-layer core-shell structure nanoparticles(NPs)could serves as carriers for targeting co-delivery of chemotherapeutic drug and anti-angiogenesis drugs.Tumor cells and vascular endothelial cells always coexist with each other,mutual promotion.Anti-angiogenesis therapy and radiotherapy or chemotherapy can also targeting tumor vascular endothelial cells and tumor cells.With the rapid development of nanotechnology and its combination with the medical field,this double-layer core-shell structure NPs system may represent a new method for the future treatment of cancer.Objective1.In this study we explored the feasibility of the novel prepared double-layer controlled release NPs as drug carriers for gene therapy and combined chemotherapy.2.We also clarified the role of double-layer NPs targeting tumor angiogenesis and breast cancer,and studied the mechanism of inhibition of tumor in vivo and in vitro.Methods1.Preparation and characterization of the NPs.The double-layered 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 and in vitro release were estimated.2.The cytotoxicities of the NPs in MCF-7 and HUVEC cells.The in vitro cytotoxicities effects of the NPs in thses two cells were evaluated using 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.In vivo antitumor study.The in vivo toxicity of the NPs were evaluated by acute toxicity test.The distribution of NPs in nude mice bearing MCF-7 tumor were evaluated by in vivo imaging technique.The xenograft tumor model was established by implanted MCF-7 cells subcutaneously into nude mice and further the in vivo antitumor activity of the NPs were evaluated by the xenograft tumor model.4.Preparation and characterization of the double layer NPs loaded with si RNA/EPI.The ability DNA bind to m PEG-g-CS complexes were investigated by agarose gel electrophoresis method and cell transfection experiments.The si RNA/EPI loaded doublelayer NPs on cell viability in vitro were investigated by CCK-8 kit.The efficacy of doublelayer NPs in vivo were assessed by the xenograft tumor model.Results1.The TEM images showed the double layer NPs had the regularly spherical shapes.And the particle size distribution results showed it had a relatively uniform distributions.The drug entrapment efficiency and drug loading efficiency of double-layer drug loaded NPs were improved compared with that of single-layer NPs.The two drugs loaded in double-layer NPs showed different release rate by the in vitro release.That is,the disruption of this envelope resulted in a rapid deployment of the anti-angiogenesis agent,the subsequent slow release of the cytotoxic agents from the NPs should then kill the tumour cells.2.It could be seen that these three blank NPs(double-layer NPs,PLGA NPs and m PEG-g-CS NPs)had no significant influence on the proliferation of MCF-7 and HUVEC cells in NPs concentration range of 0.5-1000 ?g/m L at 24 h or 48 h post administrations,thus it could be deduced that blank NPs had no cytotoxicity in the concentration range used in this study.The confocal images and flow cytometry results showed that the NPs were efficiently internalized by these cells.3.No significant differences in clinical signs between the experimental group and the control group were found and no mortality occurred throughout the entire course of the study.Additionally,no significant differences in body weight between the two studied groups were observed.The distribution and metabolism of drugs showed that three kinds of NPs could prolong the circulation time in vivo,had a certain target,and the effect of double-layer NPs was best.After intravenous injection to MCF-7-bearing nude mice,double-layer NPs achieved the enhanced anti-tumor and anti-angiogenic effects,the prolonged circulation time in blood,increased distributions in the tumor,and significantly reduced the microvessel density(MVD)in tumor tissues compared to both PLGA NPs and mPEG-g-CS NPs.4.The results of agarose gel electrophoresis showed that the m PEG-g-CS can effectively combine with p DNA to form stable complex.The cell transfection experiments showed that the double-layer NPs can successfully transfect in MCF-7 and HUVEC cells and express green fluorescent protein.The results of cytotoxicity test showed that the double-layer NPs could be used as a si RNA vector,and the NPs showed a good anti-tumor effect in vitro.In this current study,an proper transfection ratio of N/P(20:1)and transfection concentration of si RNA(100 n M)was used.The results of in vivo anti-tumor activity showed that the treatment group showed a certain anti-tumor effect,compared with had a more significant anti-tumor effect.Obviously,the double layer NPs combination of gene therapy and chemotherapy significantly inhibited the tumor growth and the average tumor size was markedly smaller than those of the control group and the treatment groups of single layer NPs.Conclusion1.In this study,the double-layer NPs showed excellent sustained release properties both in vitro and in vivo,long cycle and tumor targeting.The novel prepared double-layer controlled release NPs can be drug carriers for gene therapy and combined chemotherapy.2.This novel NPs system represents a new way of combining traditional combination chemotherapy and anti-angiogenesis gene therapy for controlled drug delivery applications,and exhibits advantages for the combination therapy against breast cancer.