C225偶联磁性白蛋白纳米球的制备、表征及联合磁流体热疗体外靶向抗肺癌的研究

Preparation and characterization of C225 conjugated magnetic albumin nanoparticles and targeted anti-lung cancer in vitro when combined magnetic fluid hyperthermia

目的:制备抗表皮生长因子受体(epithelial growth factor receptor,EGFR)单抗西妥昔(C225)偶联的磁性白蛋白纳米球,对其表征并联合磁流体热疗观察其体外抑制肺癌细胞的效果。方法:化学共沉淀法制备Fe3O4磁性纳米粒,去溶剂化-交联法制备磁性白蛋白纳米球(magnetic albumin nanospheres,MANs),然后用双功能交联剂N-琥珀酰亚胺-3-(2-吡啶二硫代)丙酸酯[N-succinimidyl-3-(2-pyridyldithio)propionate,SPDP]将C225偶联其上制备C225-MANs。纳米球的形态、平均粒径、zeta电位、抗体结合率、含铁量、细胞靶向性及磁热动力学均被表征。最后采用CCK-8及流式细胞(flow cytometry,FCM)分析检测C225-MANs介导的体外双靶向磁流体热治疗肺腺癌细胞GLC-82的效果。结果:透射电镜显示制备的C225-MANs为球形,Fe3O4纳米粒被封装其内,Fe含量约2.0 mg/mL;平均水合粒径约为140 nm,带负电;在输出电流20 A、输出频率200 kHz的交变磁场作用下,不同Fe含量的C225-MANs能在60 min内升温到39.3℃～52.0℃,且加热30 min后温度能保持恒定。FCM荧光检测显示抗体结合率为79.74%。免疫荧光实验和细胞MRI成像证实C225-MANs能够靶向GLC-82细胞。CCK-8与FCM分析显示双靶向联合治疗组的细胞增殖率明显低于其他治疗组,而凋亡率明显高于其他治疗组(P <0.05)。结论:C225-MANs对肺腺癌细胞GLC-82具有良好的靶向效应,且双靶向磁流体热疗联合分子靶向治疗在体外能有效抑制肺癌细胞。

Objective:To prepare and characterize magnetic albumin nanospheres conjugated with cetuximab (C225),which is anti- epithelial growth factor receptor (EGFR)monoclonal antibody,as well as their in vitro anti-lung cancer effect when combined with magnetic fluid hyperthermia.Methods:Fe304nanoparticles (NPs)were prepared by chemical co-precipitation,and magnetic albumin nanospheres (MANs)were prepare by desolvation-crosslinking method,then C225was further conjugated to synthesize C225-MANs using N -succinimidyl -3-(2-pyridyldithio)propionate (SPDP).Their morphology,mean particle size,zeta potential,the antibody conjugation efficiency,iron content,specific cell-binding ability,and magnetothermal dynamic profiles were characterized.Lastly,the therapeutic effect of C225-MANs induced double targeted magnetic fluid hyperthermia on lung adenocarcinoma in vitro was evaluated using CCK-8and flow cytometry (FCM)assay.Results:TEM micrographs showed that the prepared C225-MANs were approximately spherical.Fe304NPs were well incorporated into the core of the nanospheres and the iron content was about 2.0mg/mL.The mean hydrodynamic diameter was about 140nm with negative charge.When placing in alternating magnetic field with the output frequency of 200kHz and the output current of 20A,the corresponding C225-MANs with different Fe concentrations could rise to a steady temperature ranging from 39.3℃ to 52.0℃,and the temperature could keep stable without any change after 30min heating.The fluorescence percentage detected by FCM analysis revealed that the antibody conjugation efficiency was 79.74%.The ability of nanospheres targeting GLC-82cells in vitro was confirmed by immunofluoreseence experiments and magnetic resonance imaging.The proliferation rate of double C225and magnetic-targeted combined group was significantly lower than any other therapy group (P < 0.05),while the apoptotic rate of the double C225and magnetic-targeted combined treatment group were significantly higher than any other therapy group (P <0.05).Conclusion:C225-MANs had good targeting effect on GLC-82cells at cellular level.Moreover,dual- targeted magnetic fluid hyperthermia combined with molecular targeted therapy can effectively combat lung cancer in vitro.