CAS OpenIR  > 中科院上海应用物理研究所2004-2010年
磁靶向纳米药物载体的制备工艺方法学研究及在免疫分析中的初步应用
Alternative Titlethe Preparation method of magnetic targeted nano-drug carrier and its application in immuno-analyse
梁胜
Subtype博士
Thesis Advisor汪勇先
2007-01-18
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral上海应用物理研究所
Keyword磁性纳米微粒 磁性分离 Hepama-1 188re 放射性标记 靶向治疗
Abstract目的: 研制通用型用于放免分析的纳米级磁性固相分离载体以及构建具有肿瘤靶向特异性的188Re标记的磁-生物双靶向载药系统,探索其作为肿瘤导向治疗及磁靶向治疗双重治疗药物的可行性。 方法: [1] 采用化学共沉淀法合成Fe3O4磁性纳米微粒及水相/油相磁流体,考察了不同的分散剂对其稳定性的影响。 [2] 对所制备的磁性纳米微粒进行了表面修饰,使其具有不同的功能团,将针对肝癌基因表达蛋白的人源化改造单抗Hepama-1与磁性纳米微粒进行连接,构建免疫磁性纳米微粒。 [3] 采用直接标记法将188Re标记到Hepama-1及免疫磁性纳米微粒上并对其标记条件进行优化,对其标记原理进行了探索;采用羰基铼标记法,以fac-[188Re(CO)3(H2O)3]+作为放射性标记前体,二(2-吡啶甲基)-氨基]-乙酸(PADA)为螯合剂对Hepama-1及免疫磁性纳米微粒进行标记;并测定其质量控制指标。 [4] 应用MTT法测定188Re标记的免疫磁性纳米微粒以及188Re-Hepama-1体外杀伤肝癌细胞株SMMC-7721的作用。 [5] 以氨基硅烷化磁性纳米微粒为核心化学偶联羊抗兔/驴抗兔血清/羊抗人IgG,对其活化方式及偶联效率进行了研究。 [6] 所制备的磁颗粒固相二抗作为磁分离载体,对几种放免及免放药盒中的分离试剂进行了替代和对比。 结果: 化学共沉淀法制得的Fe3O4平均粒径约为10~20nm,选用月桂酸作为分散剂,能使水相/油相磁流体保持较好的稳定性。磁性纳米微粒通过化学偶联二抗后作为磁性分离剂其各项指标达到了国外同类产品的技术要求,可用于放免分析。188Re对Hepama-1、(免疫)磁性纳米微粒的标记率均大于90%,在小牛血清中具有良好的体外稳定性,并且单抗保持较高的免疫活性。体外细胞毒性结果提示,Fe3O4纳米颗粒进入细胞后位于细胞质的溶酶体及吞噬泡内,未见进入细胞核;188Re标记免疫磁性纳米微粒能有效杀伤肝癌细胞株SMMC-7721,且呈剂量依赖性。 结论 以月桂酸为分散剂,通过化学共沉淀法制备的四氧化三铁磁流体分散性较好,磁流体制备合成工艺得到了完善。羊抗兔IgG可通过化学偶联的方法固定在磁性纳米微粒上,188Re标记Hepama-1及免疫磁性纳米微粒对肝癌基因高表达的肿瘤具有良好的靶向结合性及杀伤作用。
Other AbstractObjective To prepare Superparamagnetic Iron Oxide Nanoparticles (SPIONs) for solid separation in radioimmunoassay (RIA) with universal application and Hepama-1-coated SPIONs labeled with rhenium-188 (188Re) for the purpose of bio-magnetically targeted radiotherapy, and to evaluate its feasibility as binary therapeutic drug with magnetic targeting and radioimmunotherapy (RIT) for malignant tumor. Methods [1] SPIONs (Fe3O4) were synthesized by chemical coprecipitation of Fe2+ and Fe3+ ions ,water/oil phase magnetic fluids were prepared based on SPIONs and the influence of dispersant on stability was observed. [2] The SPIONs prepared above were chemically modified and several functional groups were introduced on its surface. Hepama-1, a well-proved monoclonal antibody directed against the liver cancer, was coated to functional magnetic nanoparticles by the cross-linker with glutaraldehyde to prepare immuno-magnetic nanoparticles (IMN). [3] A direct labeling method was adopted to radiolabel Hepama-1 and IMN with rhenium-188 and an indirect labeling method was adopted with rhenium-188 by means of fac-[188Re(CO)3(H2O)3]+ as a precursor while PADA as a chelator. The parameters of quality control such as the radiolabeling efficiency, the immunoreactivity and the in vitro stability were determined. [4] MTT colorimetric assay was used for determining in vitro cytotoxicities of 188Re labeled magnetic nanoparticle and IMN to SMMC-7721 liver cancer cell lines. [5] The sheep anti-rabbit/donkey anti-rabbit IgG were immobilized onto the magnetic nanoparticles by chemical conjugation and the coupling efficiency was studied. [6] The complex of the magnetic nanoparticles and the drug anti-rabbit IgG (magnetic nanoparticle second antibody) was then used as magnetic separation carrier for RIA. Results The average diameter of Fe3O4 nanoparticle and magnetic nanoparticle second antibody is 10~20nm and about 1000nm, respectively. The second antibody labeled magnetic nanoparticle can be used in RIA. The radiolabeling efficiency with above 90% was achieved for 188Re labeled Hepama-1, magnetic nanoparticle and IMN. 188Re labels were stable in calf serum. The better immunoreactivity was retained for 188Re labeled Hepama-1 and IMN. 188Re labeled IMN could markedly kill SMMC-7721 cells with dose dependence. Conclusions The IgG and antibody can be immobilized to magnetic nanoparticles by chemical conjugation. 188Re labeled Hepama-1 and IMN have a good character for in vivo and in vitro specific targeting to bind and kill tumor cells
Pages137
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7171
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
梁胜. 磁靶向纳米药物载体的制备工艺方法学研究及在免疫分析中的初步应用[D]. 上海应用物理研究所. 中国科学院上海应用物理研究所,2007.
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