pH/氧化还原双敏感聚合物的合成与siRNA复合胶束的制剂学性质
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摘要
目的合成聚乙二醇-聚乳酸-聚组氨酸-聚乙烯亚胺(methoxy-poly(ethylene glycol)-polylactide-polyhistidine-ss-polyethylenimine,mPEG-PLA-PHis-ss-PEI)阳离子聚合物并制备siRNA胶束复合物,考察其体外制剂学性质。方法以聚乙二醇单甲醚、D,L-丙交酯、聚组氨酸和聚乙烯亚胺为起始原料,合成聚乙二醇-聚乳酸-聚组氨酸-聚乙烯亚胺,并应用核磁共振氢谱和凝胶渗透色谱进行表征;采用薄膜分散法制备mPEG-PLA-PHis-ss-PEI/siRNA胶束复合物(micelleplexes);动态激光散射法测定胶束复合物的粒径和Zeta电位值;透射电镜观察胶束复合物的形态;超速离心法测定胶束复合物中,小干扰RNA(small interference RNA,siRNA)的包封率;琼脂糖凝胶电泳阻滞法分别考察阳离子聚合物抵御阴离子置换能力、血清稳定性和胶束复合物在弱酸性和还原性条件下siRNA的触发释放行为。结果所制备的胶束复合物呈类球形,大小均一,分布均匀;当氮磷比(nitrogen/phosphoruse molar ratio,N/P)为20时,胶束复合物平均粒径为104.0 nm,Zeta电位值为30.1 mV,siRNA的包封率为(92.9±2.4)%;凝胶电泳阻滞实验结果显示,当N/P大于7时,胶束复合物具有良好的阴离子抵御能力及较强的血清稳定性;胶束复合物在较低N/P条件下具有pH敏感和还原敏感性。结论所制备的胶束复合物稳定性较好,可用于递送siRNA。
Objective To synthesize and characterize methoxy-poly(ethylene glycol)-polylactide-polyhistidine-ss-polyethylenimine(mPEG-PLA-PHis-ss-PEI)cationic micelleplexes.Methods Cationic coplolymer mPEG-PLA-PHis-ss-PEI was synthesized by ring-opening polymerization and its chemical structure was verified by 1 H NMR.The thin film dispersion method and electrostatic interaction were used to prepare small interference RNA(siRNA)based micelleplexes.Dynamic light scattering method was used to determine the particle size and Zeta potential of micelleplexes.The morphology of micelleplexes was observed using transmission electron microscope.The entrapment efficiency of siRNA in micelleplexes was determined by centrifugal ultrafiltration.The agarose gel retardation assay was used to evaluate the binding ability in micelleplexes,resistance to heparin replacement of micelleplexes,the ability to protect siRNA against RNase degradation of micelleplexes and investigate the release of siRNA by the disassembly of micelleplexes in low pH and reductive conditions.Results The micelleplexes had round shape and uniform dispersity.When N/P was 20,the particle size of the micelleplexes was 104.0 nm,Zeta potential was 30.1 mV,the entrapment efficiency of siRNA was(92.9±2.4)%,respectively.When the N/P reached 7,the micelleplexes exhibited a strong binding ability with siRNA.The micelleplexes also had strong resistance to heparin replacement and the good ability to protect siRNA against RNase degradation;the miclleplexes showed excellent pH and redox responsive behaviors.Conclusion The prepared micelleplexes loaded with siRNA show good in vitro properties and have great potentials for siRNA delivery.
Objective To synthesize and characterize methoxy-poly(ethylene glycol)-polylactide-polyhistidine-ss-polyethylenimine(mPEG-PLA-PHis-ss-PEI)cationic micelleplexes.Methods Cationic coplolymer mPEG-PLA-PHis-ss-PEI was synthesized by ring-opening polymerization and its chemical structure was verified by 1 H NMR.The thin film dispersion method and electrostatic interaction were used to prepare small interference RNA(siRNA)based micelleplexes.Dynamic light scattering method was used to determine the particle size and Zeta potential of micelleplexes.The morphology of micelleplexes was observed using transmission electron microscope.The entrapment efficiency of siRNA in micelleplexes was determined by centrifugal ultrafiltration.The agarose gel retardation assay was used to evaluate the binding ability in micelleplexes,resistance to heparin replacement of micelleplexes,the ability to protect siRNA against RNase degradation of micelleplexes and investigate the release of siRNA by the disassembly of micelleplexes in low pH and reductive conditions.Results The micelleplexes had round shape and uniform dispersity.When N/P was 20,the particle size of the micelleplexes was 104.0 nm,Zeta potential was 30.1 mV,the entrapment efficiency of siRNA was(92.9±2.4)%,respectively.When the N/P reached 7,the micelleplexes exhibited a strong binding ability with siRNA.The micelleplexes also had strong resistance to heparin replacement and the good ability to protect siRNA against RNase degradation;the miclleplexes showed excellent pH and redox responsive behaviors.Conclusion The prepared micelleplexes loaded with siRNA show good in vitro properties and have great potentials for siRNA delivery.
引文
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[2] CARTHEW R W,SONTHEIMER E J.Origins and Mechanisms of miRNAs and siRNAs [J].Cell,2009,136(4):642-655.
[3] WOLFF J A,ROZEMA D B.Breaking the bonds:non-viral vectors become chemically dynamic [J].Mol Ther,2008,16(1):8-15.
[4] KOLLI S,WONG S-P,HARBOTTLE R,et al.pH-Triggered nanoparticle mediated delivery of siRNA to liver cells in vitro and in vivo [J].Bioconjugate Chem,2013,24(3):314-332.
[5] ZHU J,QIAO M,WANG Q,et al.Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA [J].Biomaterials,2018,162:47-59.
[6] 沈雁,涂家生,庞卉,等.凝胶电泳法及荧光光度法测定siRNA阳离子脂质体的含量和包封率[J].药学学报,2009,44(04):430-435.
[7] PINEIRO L,NOVO M,AL-SOUFI W.Fluorescence emission of pyrene in surfactant solutions [J].Adv Colloid Interfac,2015,215:1-12.
[8] LIU R,LI D,HE B,et al.Anti-tumor drug delivery of pH-sensitive poly(ethylene glycol)-poly(L-histidine-)-poly(L-lactide)nanoparticles [J].Journal of Controlled Release,2011,152(1):49-56.
[9] CHIPER M,TOUNSI N,KOLE R,et al.Self-aggregating 1.8 kDa polyethylenimines with dissolution switch at endosomal acidic pH are delivery carriers for plasmid DNA,mRNA,siRNA and exon-skipping oligonucleotides [J].Journal of Controlled Release,2017,246:60-70.
[10] WHITEHEAD K A,LANGER R,ANDERSON D G.Knocking down barriers:advances in siRNA delivery [J].Nat Rev Drug Discov,2009,8(2):129-138.