两亲性嵌段聚合物PLGA-b-(PEI-co-PEG)的合成及其自组装电正性胶束的表征
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摘要
目的合成一种生物可降解与低细胞毒性的两亲性嵌段共聚物PLGA-b-(PEI-co-PEG),并研究其胶束化行为。方法采用开环聚合法合成PLGA;使用低相对分子质量的聚乙烯亚胺(PEI1800)与聚乙二醇(PEG2000)相互交联合成水溶性PEI-co-PEG共聚物;采用脱水缩合法,合成PLGA-b-(PEI-co-PEG)。根据PEI-co-PEG在37℃PBS中孵育不同时间的相对分子质量变化情况,评估其生物降解性。通过MTT法测定PLGA-b-(PEI-co-PEG)与PEI-co-PEG对MCF-7的细胞毒性。采用标准透析法制备电正性PLGA-b-(PEI-co-PEG)胶束,使用马尔文激光粒度分析仪测定其粒径分布与Zeta电位;采用简单混合法制备PLGA-b-(PEIco-PEG)胶束/胰岛素复合物;使用透射电镜表征胶束及胶束/胰岛素复合物的形貌。采用荧光猝灭法,测定胶束/胰岛素复合物在不同浓度盐离子溶液中的稳定性。结果成功合成了两亲性嵌段共聚物PLGA-b-(PEI-co-PEG)。PEI-co-PEG在37℃PBS溶液中的降解半衰期约为48 h。PLGA-b-(PEI-co-PEG)与PEI-co-PEG对MCF-7的半数抑制浓度(IC50)分别为1375.7μg/m L与425.1μg/m L。PLGA-b-(PEI-co-PEG)胶束(粒径:99.5±2.61 nm,Zeta电位:52.9±2.38 m V)可与胰岛素形成纳米尺寸的胶束/胰岛素复合物;胶束/胰岛素复合物在150 mmol/L Na Cl溶液中的解离率为27.6%。结论 PEI-co-PEG在体外条件下展现了较好的降解性。PLGA-b-(PEI-co-PEG)的细胞毒性显著低于PEI-co-PEG(P<0.05)。PLGA-b-(PEI-co-PEG)胶束/胰岛素复合物在生理条件下具有良好的盐离子稳定性。
Objective To synthesize a biodegradable and minimally cytotoxic amphiphilic block copolymer of PLGA-b-(PEI-coPEG) and study its micellization behavior. Methods PLGA was synthesized by ring-opening polymerization. The cross-linked copolymer of PEI-co-PEG was synthesized from the low-molecular-weight polyethyleneimine(PEI, 1800 D) and hydrophilic poly(ethylene glycol)(PEG, 2000 D). PLGA-b-(PEI-co-PEG) was synthesized by dehydration condensation reaction of PLGA and water soluble PEI-co-PEG. The biodegradability of PEI-co-PEG was evaluated according to the molecular weight change after incubation at 37 ℃ for different time. The cytotoxicity of PLGA-b-(PEI-co-PEG) and PEI-co-PEG in MCF-7 cells was determined by MTT assay. The cationic PLGA-b-(PEI-co-PEG) micelles were prepared by standard dialysis method. The particle size and Zeta potential of the micelles were measured by a Malvern laser particle size analyzer. Micelle/insulin complexes were prepared by simple mixing method and their morphology were characterized by transmission electron microscopy(TEM). The fluorescence quenching method was used to determine the stability of the micelle/insulin complexes at different salt concentrations. Results Amphiphilic block copolymer of PLGA-b-(PEI-co-PEG) was successfully synthesized. The half-life of PEI-co-PEG degradation in PBS at 37 ℃ was about 48 h. The 50% cell inhibiting concentration(IC50) of PLGA-b-(PEIco-PEG) and PEI-co-PEG in MCF-7 cells were 1375.7 μg/m L and 425.1 μg/m L, respectively. The micelles of PLGA-b-(PEI-coPEG)(particle size: 99.5±2.61 nm, Zeta potential: 52.9±2.38 m V) were complexed with insulin via electrostatic interaction and formed nanoscale micelle/insulin complexes. The dissociation rate of micelle/insulin complexes in 150 mmol/L Na Cl solution was 27.6%. Conclusion The synthesized PEI-co-PEG shows good degradability in vitro. The cytotoxicity of PLGA-b-(PEI-coPEG) is significantly lower than PEI-co-PEG, and PLGA-b-(PEI-co-PEG) micelle/insulin complexes have good salt-resistant stability in physiological condition.
Objective To synthesize a biodegradable and minimally cytotoxic amphiphilic block copolymer of PLGA-b-(PEI-coPEG) and study its micellization behavior. Methods PLGA was synthesized by ring-opening polymerization. The cross-linked copolymer of PEI-co-PEG was synthesized from the low-molecular-weight polyethyleneimine(PEI, 1800 D) and hydrophilic poly(ethylene glycol)(PEG, 2000 D). PLGA-b-(PEI-co-PEG) was synthesized by dehydration condensation reaction of PLGA and water soluble PEI-co-PEG. The biodegradability of PEI-co-PEG was evaluated according to the molecular weight change after incubation at 37 ℃ for different time. The cytotoxicity of PLGA-b-(PEI-co-PEG) and PEI-co-PEG in MCF-7 cells was determined by MTT assay. The cationic PLGA-b-(PEI-co-PEG) micelles were prepared by standard dialysis method. The particle size and Zeta potential of the micelles were measured by a Malvern laser particle size analyzer. Micelle/insulin complexes were prepared by simple mixing method and their morphology were characterized by transmission electron microscopy(TEM). The fluorescence quenching method was used to determine the stability of the micelle/insulin complexes at different salt concentrations. Results Amphiphilic block copolymer of PLGA-b-(PEI-co-PEG) was successfully synthesized. The half-life of PEI-co-PEG degradation in PBS at 37 ℃ was about 48 h. The 50% cell inhibiting concentration(IC50) of PLGA-b-(PEIco-PEG) and PEI-co-PEG in MCF-7 cells were 1375.7 μg/m L and 425.1 μg/m L, respectively. The micelles of PLGA-b-(PEI-coPEG)(particle size: 99.5±2.61 nm, Zeta potential: 52.9±2.38 m V) were complexed with insulin via electrostatic interaction and formed nanoscale micelle/insulin complexes. The dissociation rate of micelle/insulin complexes in 150 mmol/L Na Cl solution was 27.6%. Conclusion The synthesized PEI-co-PEG shows good degradability in vitro. The cytotoxicity of PLGA-b-(PEI-coPEG) is significantly lower than PEI-co-PEG, and PLGA-b-(PEI-co-PEG) micelle/insulin complexes have good salt-resistant stability in physiological condition.
引文
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[26]Wong CY,Al-Salami H,Dass CR.Potential of insulin nanoparticle formulations for oral delivery and diabetes treatment[J].J Control Release,2017,264:247-75.
[27]Mishra D,Kang HC,Bae YH.Reconstitutable charged polymeric(PLGA)(2)-b-PEI micelles for gene therapeutics delivery[J].Biomaterials,2011,32(15):3845-54.
[2]Anderson JM,Shive MS.Biodegradation and biocompatibility of PLA and PLGA microspheres[J].Adv Drug Deliv Rev,2012,64(S):72-82.
[3]Mir M,Ahmed N,Rehman AU.Recent applications of PLGA based nanostructures in drug delivery[J].Colloids Surf B Biointerfaces,2017,159:217-31.
[4]Yun YH,Lee BK,Park K.Controlled drug delivery:historical perspective for the next generation[J].J Control Release,2015,219:2-7.
[5]Ungaro F,D'angelo I,Miro A,et al.Engineered PLGA nano-and micro-carriers for pulmonary delivery:challenges and promises[J].J Pharm Pharmacol,2012,64(9):1217-35.
[6]Xu Y,Kim CS,Saylor DM,et al.Polymer degradation and drug delivery in PLGA-based drug-polymer applications:A review of experiments and theories[J].J Biomed Mater Res B Appl Biomater,2017,105(6):1692-716.
[7]Park JH,Kang HJ,Kwon DY,et al.Biodegradable poly(lactide-coglycolide-co-epsilon-caprolactone)block copolymers-evaluation as drug carriers for a localized and sustained delivery system[J].J Mater Chem B,2015,3(41):8143-53.
[8]Wang X,Wenk E,Hu X,et al.Silk coatings on PLGA and alginate microspheres for protein delivery[J].Biomaterials,2007,28(28):4161-9.
[9]Lim HP,Tey BT,Chan ES.Particle designs for the stabilization and controlled-delivery of protein drugs by biopolymers:a case study on insulin[J].J Control Release,2014,186:11-21.
[10]Kang HC,Lee JE,Bae YH.Nanoscaled buffering zone of charged(PLGA)n-b-b PEI micein acidic microclimate for potential protein delivery application[J].J Control Release,2012,160(3):440-50.
[11]Park W,Kim D,Kang HC,et al.Multi-arm histidine copolymer for controlled release of insulin from poly(lactide-co-glycolide)microsphere[J].Biomaterials,2012,33(34):8848-57.
[12]Ahn CH,Chae SY,Bae YH,et al.Biodegradable poly(ethylenimine)for plasmid DNA delivery[J].J Control Release,2002,80(1/3):273-82.
[13]Chen Y,Yang Z,Liu C,et al.Synthesis,characterization,and evaluation of paclitaxel loaded in six-arm star-shaped poly(lactic-coglycolic acid)[J].Int J Nanomedicine,2013,8:4315-26.
[14]Mei FF,Peng Y,Lu ST,et al.Synthesis and characterization of biodegradable poly(lactic-co-glycolic acid)[J].J Macromol Sci,Part B:Physics,2015,54(5):562-70.
[15]Gou PF,Zhu WP,Xu N,et al.Synthesis and characterization of welldefined cyclodextrin-centered seven-arm star poly(epsilon-caprolactone)s and amphiphilic star poly(epsilon-caprolactone-b-ethylene glycol)s[J].J Polym Sci A Polym Chem,2008,46(19):6455-65.
[16]Park MR,Han KO,Han IK,et al.Degradable polyethylenimine-altpoly(ethylene glycol)copolymers as novel gene carriers[J].J Control Release,2005,105(3):367-80.
[17]Jacob D,Joan Taylor M,Tomlins P,et al.Synthesis and identification of FITC-insulin conjugates produced using human insulin and insulin analogues for biomedical applications[J].J Fluoresc,2016,26(2):617-29.
[18]Park W,Park SJ,Na K.The controlled photoactivity of nanoparticles derived from Ionic interactions between a water soluble polymeric photosensitizer and polysaccharide quencher[J].Biomaterials,2011,32(32):8261-70.
[19]郭圣荣.药用高分子材料[M].北京:人民卫生出版社,2009:390-1.
[20]Sawant RR,Sriraman SK,Navarro G,et al.Polyethyleneimine-lipid conjugate-based p H-sensitive micellar carrier for gene delivery[J].Biomaterials,2012,33(15):3942-51.
[21]Tang GP,Zeng JM,Gao SJ,et al.Polyethylene glycol modified polyethylenimine for improved CNS gene transfer:effects of PEGylation extent[J].Biomaterials,2003,24(13):2351-62.
[22]Endres TK,Beck-Broichsitter M,Samsonova OA,et al.Selfassembled biodegradable amphiphilic PEG-PCL-l PEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery[J].Biomaterials,2011,32(30):7721-31.
[23]Lv J,Yang J,Hao XE,et al.Biodegradable PEI modified complex micelles as gene carriers with tunable gene transfection efficiency for ECs[J].J Mater Chem B,2016,4(5):997-1008.
[24]Ramezani M,Ebrahimian M,Hashemi M.Current strategies in the modification of PLGA-based gene delivery system[J].Curr Med Chem,2017,24(7):728-39.
[25]Yin L,Yuvienco C,Montclare JK.Protein based therapeutic delivery agents:Contemporary developments and challenges[J].Biomaterials,2017,134:91-116.
[26]Wong CY,Al-Salami H,Dass CR.Potential of insulin nanoparticle formulations for oral delivery and diabetes treatment[J].J Control Release,2017,264:247-75.
[27]Mishra D,Kang HC,Bae YH.Reconstitutable charged polymeric(PLGA)(2)-b-PEI micelles for gene therapeutics delivery[J].Biomaterials,2011,32(15):3845-54.