聚乙二醇接枝率对第五代聚酰胺-胺树状大分子体外毒性与细胞摄取的影响
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摘要
目的研究聚乙二醇(PEG)不同接枝率对第五代聚酰胺-胺(polyanfido-amine,PAMAM G5)树状大分子体外毒性与细胞摄取的影响。方法合成4种不同接枝率的PEG-PAMAM G5,采用核磁共振氢谱(~1H-NMR)、红外光谱(FT-IR)、纳米粒度-电位分析仪进行结构鉴定和表征,溶血毒性和细胞毒性实验考察体外安全性,细胞摄取实验及激光共聚焦显微镜考察摄取情况及胞内定位。结果 4种PEG-PAMAM G5的PEG接枝率分别为7.8%,14.1%,20.3%和24.2%,粒径依次为(17.05±1.77)nm<(20.77±1.02)nm<(21.68±1.04)nm<(23.19±0.54)nm,Zeta电位由PAMAM G5的(25.57±1.37)mV降低至PEG_(31)-PAMAM G5的(9.27±0.40)mV。PAMAM G5经PEG修饰后,溶血毒性、细胞毒性和细胞摄取均显著降低(P<0.05),仍可被HBMEC细胞摄取并进入细胞核。结论 PEG修饰可显著降低PAMAM G5的体外细胞毒性,随着PEG接枝率的增大,Zeta电位降低,减毒效果越显著;经修饰后的PEG-PAMAM G5仍具有进入细胞核的能力。因此,PEG化树状大分子可作为基因或细胞核靶向药物的载体进行更深入的研究。
OBJECTIVE To study the effect of different graft ratios of PEG on the toxicity in vitro and cellular uptake of PAMAM G5 dendrimers.METHODS Nuclear magnetic resonance(~1H-NMR) and Fourier transform infrared(FT-IR) spectroscopy were used to confirm the structure of PEG-PAMAM G5 dendrimers with four different graft ratios.The particle size and Zeta potential of the nanoparticles were determined by nanoparticle size-Zeta potential analyzer.The toxicity in vitro,cellular uptake,and intracellular localization were tested by hemolysis assay,cytotoxicity assay,cellular uptake test,and laser scanning confocal microscope images,respectively.RESULTS The particle sizes of dendrimers with PEG graft ratios of 7.8%,14.1%,20.3%,and 24.2%were(17.05 ±1.77),(20.77±1.02),(21.68 ± 1.04),and(23.19 ± 0.54) nm,respectively.The Zeta potential decreased from(25.57 ± 1.37) mV of PAMAM G5 to(9.27 ± 0.40) mV of PEG_(31)-PAMAM G5.In addition,the hemolytic toxicity and cytotoxicity of PAMAM G5 dendrimers also markedly decreased especially at high concentrations because of PEG modification.Moreover,the PEG-PAMAM G5 dendrimers with particle diameter of nearly 20 nm not only could be taken in by HBMEC cells,but also accumulated in the cell nucleus.CONCLUSION Modification of PEG can greatly reduce the toxicity of PAMAM G5 dendrimers in vitro,and the higher the degree of modification,the more obvious is the attenuated effect.The PEG-PAMAM G5 dendrimers with particle diameter larger than 20 nm still can be taken in by HBMEC cells and accumulate in the cell nucleus,which provide a foundation for the further research using modified PEG-PAMAM G5 as a basic carrier for genes and nuclear targeting agents in nano medicine.
OBJECTIVE To study the effect of different graft ratios of PEG on the toxicity in vitro and cellular uptake of PAMAM G5 dendrimers.METHODS Nuclear magnetic resonance(~1H-NMR) and Fourier transform infrared(FT-IR) spectroscopy were used to confirm the structure of PEG-PAMAM G5 dendrimers with four different graft ratios.The particle size and Zeta potential of the nanoparticles were determined by nanoparticle size-Zeta potential analyzer.The toxicity in vitro,cellular uptake,and intracellular localization were tested by hemolysis assay,cytotoxicity assay,cellular uptake test,and laser scanning confocal microscope images,respectively.RESULTS The particle sizes of dendrimers with PEG graft ratios of 7.8%,14.1%,20.3%,and 24.2%were(17.05 ±1.77),(20.77±1.02),(21.68 ± 1.04),and(23.19 ± 0.54) nm,respectively.The Zeta potential decreased from(25.57 ± 1.37) mV of PAMAM G5 to(9.27 ± 0.40) mV of PEG_(31)-PAMAM G5.In addition,the hemolytic toxicity and cytotoxicity of PAMAM G5 dendrimers also markedly decreased especially at high concentrations because of PEG modification.Moreover,the PEG-PAMAM G5 dendrimers with particle diameter of nearly 20 nm not only could be taken in by HBMEC cells,but also accumulated in the cell nucleus.CONCLUSION Modification of PEG can greatly reduce the toxicity of PAMAM G5 dendrimers in vitro,and the higher the degree of modification,the more obvious is the attenuated effect.The PEG-PAMAM G5 dendrimers with particle diameter larger than 20 nm still can be taken in by HBMEC cells and accumulate in the cell nucleus,which provide a foundation for the further research using modified PEG-PAMAM G5 as a basic carrier for genes and nuclear targeting agents in nano medicine.
引文
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[19]MA K,HU M X,QI Y,et al.PAMAM-Triamcinolone acetonide conjugate as a nucleus-targetinggene carrier for enhanced transfer activity[J].Biomaterials,2009,30(30):6109-6118.
[20]DAVIS M E,CHEN Z G,SHIN D M.Nanoparticle therapeutics:An emerging treatment modality for cancer[J].Nat Rev Drug Discov,2008,7(9):771-782.
[2]JEDRYCH M,BOROWSKA K,GALUS R,et al.The evaluation of the biomedical effectiveness of poly(amido)amine dendrimers generation 4.0 as a drug and as drug carriers:a systematic review and Meta-analysis[J].Int J Pharm,2014,462(1-2):38-43.
[3]POURIANAZAR N T,MUTLU P,GUNDUZ U.Bioapplications of poly(amidoamine)(PAMAM)dendrimers in nanomedicine[J].J Nanopart Res,2014,16(4):1-38.
[4]BAI S,AHSAN F.Synthesis and evaluation of pegylateddendrimericnanocarrier for pulmonary delivery of low molecular weight heparin[J].Pharm Res,2009,26(3):539-548.
[5]ESFAND R,TOMALIA D A.Poly(amidoamine)(PAMAM)dendrimers:from biomimicry to drug delivery and biomedical applications[J].Drug Discov Today,2001,6(8):427-436.
[6]TSUTSUMI T,HIRAYAMA F,UEKAMA K,et al.Evaluation of polyamidoamine dendrimer/alpha-cyclodextrin conjugate(generation 3,G3)as a novel carrier for small interfering RNA(siRNA)[J].J Controlled Release,2007,119(3):349-359.
[7]SANTOS J L,OLIVEIRA H,PANDITA D,et al.Functionalization of poly(amidoamine)dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells[J].J Controlled Release,2010,144(1):55-64.
[8]BAI S,THOMAS C,RAWAT A,et al.Recent progress in dendrimer-based nanocarriers[J].Crit Rev Ther Drug Carrier Syst,2006,23(6):437-495.
[9]HUANG S X,LI J F,HAN L,et al.Dual targeting effect of angiopep-2-modified,DNA-loaded nanoparticles for glioma[J].Biomaterials,2011,32(28):6832-6838.
[10]WANG K,ZHANG X F,LIU Y,et al.Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicinpolymer conjugates[J].Biomaterials,2014,35(30):8735-8747.
[11]ODDONE N,ZAMBRANA A I,TASSANO M,et al.Cell uptake mechanisms of PAMAM G4-FITC dendrimer in human myometrial cells[J].J Nanopar Res,2013,15(7):1-14.
[12]PENG J Q,QI X L,CHEN Y,et al.Octreotide-conjugated PAMAM for targeted delivery to somatostatin receptors over-expressed tumor cells[J].J Drug Target,2014,22(5):428-438.
[13]HE H,LI Y,JIA X R,et al.PEGylated poly(amidoamine)dendrimer-based dual-targeting carrier for treating brain tumors[J].Biomaterials,2011,32(2):478-487.
[14]LUO D,HAVERSTICK K,BELCHEVA N,et al.Poly(ethylene glycol)-conjugated PAMAM dendrimer for biocompatible,highefficiency DNA delivery[J].Macromolecules,2002,35(9):3456-3462.
[15]NEL A E,MDLER L,VELEGOL D,et al.Understanding biophysicochemical interactions at the nano-bio interface[J].Nat Mater,2009,8(7):543-557.
[16]MOGHIMI S M.The effect of methoxy-PEG chain length and molecular architecture on lymph node targeting of immuno-PEG liposomes[J].Biomaterials,2006,27(1):136-144.
[17]TAMMAM S N,AZZAZY H M,BREITINGER H G,et al.Chitosan nanoparticles for nuclear targeting:The effect of nanoparticle size and nuclear localization sequence density[J].Mol Pharm,2015,12(12):4277-4289.
[18]GERACE L.Molecular trafficking across the nuclear pore complex[J].Curr Opin Cell Biol,1992,4(4):637-645.
[19]MA K,HU M X,QI Y,et al.PAMAM-Triamcinolone acetonide conjugate as a nucleus-targetinggene carrier for enhanced transfer activity[J].Biomaterials,2009,30(30):6109-6118.
[20]DAVIS M E,CHEN Z G,SHIN D M.Nanoparticle therapeutics:An emerging treatment modality for cancer[J].Nat Rev Drug Discov,2008,7(9):771-782.