Preparation And Characterization Of Novel Nano Composite BMs/pHSP-shPLK1/DOX Mediated By Magnetosomes Of Magnetotactic Bacteria

Objective To construct the eukaryotic expression plasmid of a target knock down Polo like protein kinase 1(PLK1)driven by heat shock promoter and observe its effect on the proliferation of osteosarcoma U2 OS cells.Methods Firstly,the promoter of heat shock protein HSP70 was synthesized.Then promoter HSP70 was cloned into the eukaryotic expression plasmid p EGFP-C1 with green fluorescent protein(GFP)tag,Replace the original CMV promoter with the heat shock promoter(p HSP),to construct eukaryotic expression vector p HSP-GFP,secondly,RNAi was used to synthesize the sh RNA sequence of PLK1 Gene,And it was cloned into the eukaryotic expression vector p HSP-GFP to form the recombinant plasmid p HSP-sh PLK1-GFP;In addition,we designed the negative control plasmid p HSP-NC-GFP.Finally,the plasmid was transfected into U2 OS cells by Lipo2000,Non heat stimulated group were cultured in normal condition,heat shock group and negative control group was heated at 42? for 2 hours,The expression of GFP was detected by immunofluorescence staining,so the driving ability of heat shock promoter was determined,The expression of PLK1 in m RNA was detected by Quantitative real-time PCR method,Western blotting was used to detect the expression level of PLK1 protein and the effect of recombinant plasmid on the proliferation of U2 OS cells was detected by MTT method.Results The eukaryotic expression plasmid p HSP-sh PLK1-GFP driven by heat shock promoter was successfully constructed,Cell immunofluorescence staining showed that the heat shock promoter could drive the expression of GFP,GFP protein is expressed in cytoplasm.The results of quantitative real-time PCR showed that the expression of PLK1 in heat shock group was significantly lower than that in non heat stimulated group and negative control group(P < 0.05).The Western blotting results showed that the expression of PLK1 protein in heat shock group was lower than that of non heat stimulated group and negative control group(P<0.05);MTT results showed that the cell proliferation activity was significantly inhibited,compared with the non heat stimulated group and negative control group(P < 0.05).Conclusion The eukaryotic expression plasmid p HSP-sh PLK1-GFP driven by heat shock promoter can be expressed in U2 OS cells,the recombinant plasmid showed a stronger down regulation of PLK1 expression and inhibition of proliferation under the heating condition of 42?.Objective Bacterial magnetosomes(BMs)as drug carriers,the eukaryotic expression plasmid p HSP-sh PLK1-GFP regulated by heat shock promoter(p HSP)and chemotherapy drugs doxorubicin(DOX)were coupled to the carrier material.Prepare the novel nano composite BMs/p HSP-sh PLK1/DOX and its characterization and transfection efficiency were detected.Methods Optimization of culture conditions for the AMB-1 by changing the type and quantity of iron,carbon and nitrogen sources in the culture medium,Separation and purification of magnetosomes by ultrasonic and magnetic adsorption.The morphological characteristics of the magnetosomes were observed under transmission electron microscope(TEM).The recombinant plasmid p HSP-sh PLK1-GFP and DOX were coupled to the magnetic body to construct the complex by using polyethylenimine(PEI).Laser particle size analyzer and Zeta potential analyzer were used to detect the particle size and the charge of the composites.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 U2 OS cells was detected by immunofluorescence assay after treatment of the complex BMs/p HSP-sh PLK1/DOX,with this method,The uptake of the complex was analyzed.Results The optimal culture condition of AMB-1 are 20?mol/L of ferrous sulfate,800 mg/L of sodium nitrate,and 200 mg/L of succinic acid.Transmission electron microscopy showed that the magnetosomes purified were of uniform size and good dispersion.The diameters and the zeta potentials of of BMs/p HSP-sh PLK1/DOX were(129.5±9.7 nm)and(-11.7±2.9 m V)m V.Under an alternating magnetic field,the complex can be heated up to 43 C in the 3min,and the magnetic field strength can be maintained 30 min by adjusting the magnetic field strength.Under these conditions,the release rate of DOX could reach 54%,and the GFP protein expression was obviously expressed in the cells treated with the complex,which confirmed that the compound could be effectively absorbed by U2 OS cell.Conclusions The compound BMs/p HSP-sh PLK1/DOX mediated by the magnetosomes is successfully prepared and the compound is expected to become a new type of nano drug for osteosarcoma treatment.