细菌磁小体共递送阿霉素和重组质粒pHSP70-Plk1-shRNA用于骨肉瘤治疗

Co-delivery of doxorubicin and recombinant plasmid pHSP70-Plk1-shRNA by bacterial magnetosomes for osteosarcoma therapy.

作者信息

Cheng Li, Ke Youqun, Yu Shuisheng, Jing Juehua

机构信息

Department of Orthopaedics, The Second Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, Anhui, People's Republic of China.

出版信息

Int J Nanomedicine. 2016 Oct 25;11:5277-5286. doi: 10.2147/IJN.S115364. eCollection 2016.

DOI:10.2147/IJN.S115364

PMID:27822032

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5087786/

Abstract

To explore a novel combination of chemotherapy, gene therapy, and thermotherapy for osteosarcoma, a targeted heat-sensitive co-delivery system based on bacterial magnetosomes (BMs) was developed. The optimal culture conditions of magnetotactic bacteria (MTB) AMB-1 and characterization of BMs were achieved. A recombinant eukaryotic plasmid heat shock protein 70-polo-like kinase 1-short hairpin RNA (pHSP70-Plk1-shRNA) under transcriptional control of a thermosensitive promoter (human HSP70 promoter) was constructed for gene therapy. Doxorubicin (DOX) and pHSP70-Plk1-shRNA were included in the targeted thermosensitive co-delivery system, and in vitro DOX release activity, targeted gene silencing efficiency and in vitro antitumor efficacy were investigated. The results showed that the optimal culture conditions of MTB AMB-1 are an oxygen concentration of 4.0%, a pH value of 7.0, 20 μmol/L of ferrous sulfate, 800 mg/L of sodium nitrate, and 200 mg/L of succinic acid. The temperature of BMs reached 43°C within 3 minutes and could be maintained for 30 minutes by adjusting the magnitude of the alternating magnetic field (AMF). The diameters of BMs, BM-DOX, BM-recombinant eukaryotic plasmid pHSP70-Plk1-shRNA (shPlk1), and BM-DOX-shPlk1 were 43.7±4.6, 79.2±5.4, 88.9±7.8, and 133.5±11.4 nm, respectively. The zeta potentials of BMs, BM-DOX, BM-shPlk1, and BM-DOX-shPlk1 were -29.4±6.9, -9.5±5.6, -16.7±4.8, and -10.3±3.1 mV, respectively. Besides, the system exhibited good release behavior. DOX release rate from BM-DOX-shPlk1 was 54% after incubation with phosphate-buffered saline at 43°C and 37% after incubation with 50% fetal bovine serum, which was significantly higher than that at 37°C (<0.05). In addition, the expressions of Plk1 mRNA and protein were significantly suppressed in cells treated with BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF compared to other groups (<0.05). Furthermore, evaluation of the effect of in vitro antitumor revealed that BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF was significantly more effective than others in tumor inhibition. In conclusion, the new heat-sensitive co-delivery system represents a promising approach for the treatment of cancer.

摘要

为探索骨肉瘤化疗、基因治疗和热疗的新型联合治疗方法，开发了一种基于细菌磁小体（BMs）的靶向热敏共递送系统。实现了趋磁细菌（MTB）AMB-1的最佳培养条件及磁小体的表征。构建了一种在热敏启动子（人HSP70启动子）转录控制下的重组真核质粒热休克蛋白70-波罗样激酶1-短发夹RNA（pHSP70-Plk1-shRNA）用于基因治疗。将阿霉素（DOX）和pHSP70-Plk1-shRNA纳入靶向热敏共递送系统，并研究了体外DOX释放活性、靶向基因沉默效率和体外抗肿瘤疗效。结果表明，MTB AMB-1的最佳培养条件为氧浓度4.0%、pH值7.0、硫酸亚铁20 μmol/L、硝酸钠800 mg/L和琥珀酸200 mg/L。磁小体在3分钟内温度达到43°C，通过调节交变磁场（AMF）强度可维持30分钟。磁小体、BM-DOX、BM-重组真核质粒pHSP70-Plk1-shRNA（shPlk1）和BM-DOX-shPlk1的直径分别为43.7±4.6、79.2±5.4、88.9±7.8和133.5±11.4 nm。磁小体、BM-DOX、BM-shPlk1和BM-DOX-shPlk1的ζ电位分别为-29.4±6.9、-9.5±5.6、-16.7±4.8和-10.3±3.1 mV。此外，该系统表现出良好的释放行为。BM-DOX-shPlk1在43°C与磷酸盐缓冲盐水孵育后的DOX释放率为54%，与50%胎牛血清孵育后的释放率为37%，显著高于37°C时的释放率（<0.05）。另外，与其他组相比，在AMF影响下热疗后用BM-DOX-shPlk1处理的细胞中Plk1 mRNA和蛋白的表达受到显著抑制（<0.05）。此外，体外抗肿瘤效果评估显示，在AMF影响下热疗后的BM-DOX-shPlk1在肿瘤抑制方面比其他组显著更有效。总之，这种新型热敏共递送系统是一种有前景的癌症治疗方法。