Objective The short hairpin RNA (shRNA) of Polo like protein kinase 1 (shPLK1) and chemotherapy drugs doxorubicin (DOX) were coupled to the carrierbacterialmagnctosomes (BMs).Prepare the novel nano composite bacterialmagnetosomes/pHSP-shPLK1/doxorubicin (BMs/pHSP-shPLK1/DOX) and its characterization were detected.Methods Optimization of culture conditions for the AMB-1 by changing composition of culture medium,Separation and purification of magnetosomes.The morphological characteristics of the magnetosomes were observed under transmission electron microscope (TEM).The recombinant plasmid pHSP-shPLK1 and DOX were coupled to the magnetosomes to construct the complex by using polyethylenimine (PEI).The particle size and the charge of the compositesweredetected.The effect of heat production and the release rate of DOX under the condition of alternating magnetic field were observed,the expression of green fluorescent protein (GFP) in osteosarcoma U2OS cells was detected by immunofluorescence assay after treatment of the complex.Results The optimal culture condition of AMB-1 are 20 μmol/L of ferrous sulfate,800 mg/L of sodium nitrate,and 200 mg/L of succinic acid.Transmission electron microscopy showed that the magnetosomes were of uniform size and good dispersion.The diameters and the zeta potentials ofof BMs/pHSP-shPLK1/DOX were (129.5 ± 9.7) nm and (-11.7 ± 2.9) mV.Under an alternating magnetic field,the complex can be heated up to 42 ℃ in the 3 min,and the magnetic field strength can be maintained 30 min.Under these conditions,In 24 h and 48 h,the content of DOX in supernatant of 50％ fatal bovine serun (FBS) group was significantly higher than that of group PBS (t24 h =6.887,P24 h =0.020;t48 h =17.145,P48 h =0.000),in 48 h the release rate of DOX could reach 54％,and the GFP protein expression was obviously expressed in the cells treated with the complex.Conclusion The compound BMs/pHSP-shPLK1/DOX mediated by the magnetosomes is successfully prepared and is expected to become a new type of nano drug for osteosarcoma treatment.%目的 将热激启动子(pHSP)调控的Polo样蛋白激酶1(shPLK1)以及柔红霉素(DOX)耦联于载体磁小体(BMs)上,制备复合物磁小体/PLK1基因/柔红霉素(BMs/pHSP-shPLK1/DOX)的干扰质粒并检测其表征.方法 改变趋磁细菌培养液中成分优化趋磁细菌AMB-1的培养条件;分离纯化磁小体,在电镜下观察磁小体的形态;利用聚乙烯亚胺(PEI)将质粒pHSP-shPLK1和DOX耦联至BMs构建复合物BMs/pHSP-shPLK1/DOX,检测复合物的粒径及电荷;观察在交变磁场(AMF)中复合物的产热效应及DOX的释放率;细胞免疫荧光检测复合物处理后U20S细胞中绿色荧光蛋白(GFP)的表达.结果 趋磁细菌AMB-1的最佳生长条件为:以20 μmol/L FeSO4为铁源,以800 mg/L NaNO3为氮源,以200 mg/L的琥珀酸为碳源;电镜显示磁小体大小均匀,分散度好;复合物BMs/pHSP-shPLK1/DOX的粒径为(129.5±9.7) nm,Zeta电位为(-11.7±2.9)mV;在AMF下,复合物可在3 min之内升温至42℃,并维持30 min,该条件下,在24 h及48 h,50％胎牛血清(FBS)组上清液中DOX含量均明显高于磷酸盐缓冲液(PBS)组(t24 h=6.887,P24 h=0.020;t48 h=17.145,P48 h =0.000),50％ FBS中DOX的释放率可达到54％;复合物处理后的细胞可见明显的GFP蛋白表达 .结论 成功制备BMs介导的复合物BMs/pHSP-shPLK1/DOX.
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