EcN共价PSMA/PELA载药微球的制备及其靶向肿瘤低氧环境的初步研究

Preparation of EcN-conjugated drug-loaded PSMA/PELA micro-particles and their ability to target hypoxic tumor environment

目的将Escherichia coli Nissle 1917(EcN)与聚苯乙烯马来酸酐(poly styrene-co-maleic anhydride, PSMA)/聚DL-乳酸-聚乙二醇共聚物[(poly (DL-Lactic acid) and poly ethyleneglycol block copolymer, PELA]聚合物载药微粒共价结合,研究其靶向肿瘤低氧环境的效果。方法溶剂挥发法制备不同混合比例(质量比为7∶3和5∶5)的PSMA和PELA携载阿霉素(Dox)的PSMA/PELA微粒;通过EDC和NHS催化反应使得EcN表面的氨基和PSMA/PELA载药微粒表面的酸酐发生共价结合,平板计数法表征接枝后EcN的生长活性;利用HepG2细胞体外构建3D肿瘤模型,研究EcN共价PSMA/PELA载药微粒在肿瘤低氧区域的渗透效果;小鼠肝癌细胞H22建立皮下肿瘤模型,体内验证其靶向肿瘤低氧区域的效果。结果成功制备载Dox的PSMA/PELA微粒(DMP_(7/3)和DMP_(5/5)),两种微粒球形规则,粒径700～800 nm;DMP_(7/3)和DMP_(5/5)载药量分别为(4.27±0.19)%和(3.94±0.11)%,包裹效率分别为(85.4±2.6)%和(78.8±3.5)%,载药量和包裹效率差异无统计学意义;CLSM结果显示,EcN成功接枝在DMP_(7/3)表面,且接枝后的细菌具有生长活力;体外渗透实验研究表明,与EcN未共价接枝的DMP比较,EcN修饰的聚合物载药微粒(EcN@DMP_(7/3))可较多地渗透至肿瘤内部;体内分布实验证实,静脉给药4 h和24 h后,EcN@DMP_(7/3)治疗组肿瘤组织内Dox浓度分别是DMP_(7/3)治疗组浓度的3倍和6倍(P<0.01)。结论通过EDC和NHS催化反应方法可使EcN表面的氨基和PSMA/PELA载药微粒表面的酸酐发生共价反应,且反应后的EcN依然维持其生长活性,能有效驱动PSMA/PELA载药微粒靶向肿瘤内部低氧区域。

Objective To conjugate Escherichia coli Nissle 1917(EcN)with drug-loaded poly styrene-co-maleic anhydride(PSMA)/poly(DL-lactic acid)and poly ethyleneglycol block copolymer(PELA)polymer micro-particles and investigate the ability of the micro-particles to target the hypoxic tumor environment.Methods PSMA and PELA were mixed at the ratio of 7∶3 or 5∶5(m/m)to prepare doxorubicin(Dox)-loaded PSMA/PELA micro-particles through solvent evaporation method.The covalent binding was achieved by EDC-NHS catalytic reaction of the carboxyl groups on the surface of EcN with the amino groups on the surface of PSMA/PELA drug-loaded micro-particles.In an in vitro 3 D tumor model constructed using HepG2 cells,we investigated the permeation of EcN-conjugated PSMA/PELA drug-loaded micro-particles into the hypoxic tumor area.We further tested the performance of the drug-loaded micro-particles for targeting the hypoxic tumor environment in a mouse model bearing subcutaneous hepatoma H22 cell xenograft.Results We successfully prepared Dox-loaded micro-particles with different PSMA/PELA ratios(DMP7/3 and DMP5/5)using solvent evaporation method,and both of the micro-particles showed a regular spherical shape with an average size of 700 to 800 nm.DMP7/3 and DMP5/5 micro-particles had comparable Dox-loading efficiency[(4.27±0.19)%vs(3.94±0.11)%]and encapsulation efficiency((85.4±2.6)%vs(78.8±3.5)%)Confocal laser scanning microscopy showed that EcN was successfully conjugated on the surface DMP7/3 micro-particles.Compared with DMP microparticles without EcN conjugation,the EcN-conjugated micro-particles(EcN@DMP7/3)permeated efficiently into the central area in the in vitro 3 D tumor model.In the tumor-bearing mice,bio-distribution analyses revealed that at 4 and 24 h after intravenous administration,EcN@DMP7/3 resulted in 3-folder and 6-folder increases in Dox accumulation in the tumors,respectively,as compared with the non-conjugated DMP7/3 micro-particles(P<0.01).Conclusion EDC-NHS catalytic reaction allows the amino groups on the outer surface of EcN to covalently bind to anhydride on the surface of drug-loaded PSMA/PELA particles without affecting the growth activity of EcN,which effectively drives the drug-loaded particles to target the hypoxic region inside the tumor.