α-CD-PAMAM抗血清阳离子聚合物的构建及其作为基因载体的性能评价
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摘要
本文合成了一种α-环糊精(α-cyclodextrin,α-CD)与聚酰胺-胺(polyamidoamine,PAMAM)的接枝聚合物(Cy D-G1)。~1H核磁测试结果表明,每个环糊精分子上平均接枝了6.4个PAMAM-G1分子。凝胶电泳结果显示,Cy D-G1可以有效结合DNA,并保护DNA免于核酸酶DNase I的降解。当载体与DNA复合物的N/P比为40时,可以压缩DNA形成平均粒径为120 nm左右的粒子,复合物表面的zeta电位约为+21 m V。该复合物可以在血清存在的条件下保持粒子的完整性并在360 min内稳定性良好。与对照品PEI-25K载体相比,Cy D-G1在高浓度时仍表现出较低的细胞毒性。将Cy D-G1与市售Lipofectamine 2000和PEI-25K对比转染发现,Cy D-G1/DNA复合物在多种细胞系中具有较高的转染率,而且转染水平不受血清影响。通过激光共聚焦观察并结合流式细胞分析表明,该阳离子聚合物介导DNA可以在4 h内有效进入细胞核内。上述结果证明,该阳离子聚合物作为一种非病毒型基因传递系统具有优良的性能以及体内给药应用的潜在可行性。
Polyamidoamine(PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer(Cy D-G1) was synthesized as a gene delivery vector. Based on ~1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that Cy D-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25 K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25 K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P = 80 compared with Lipofectamine 2000 and PEI-25 K. Unlike Lipofectamine 2000 and PEI-25 K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.
Polyamidoamine(PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer(Cy D-G1) was synthesized as a gene delivery vector. Based on ~1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that Cy D-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25 K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25 K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P = 80 compared with Lipofectamine 2000 and PEI-25 K. Unlike Lipofectamine 2000 and PEI-25 K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.
引文
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[4]Kundu PP,Sharma V.Synthetic polymeric vectors in gene therapy[J].Curr Opin Solid Sate Mater,2008,12:89-102.
[5]Thomas CE,Ehrhardt A,Kay MA.Progress and problems with the use of viral vectors for gene therapy[J].Nat Rev Genet,2003,4:346-358.
[6]Ping Y,Ma Q,Chen JH.Dextran-sperminepolycation as a vector for gene transfection in vitro[J].Acta Pharm Sin(药学学报),2007,42:669-674.
[7]Chen JH.Application of cationic polymer vector for gene delivery systems[J].Acta Pharm Sin(药学学报),2003,38:316-320.
[8]Shen M,Huang YZ,Ham LM,et al.Multifunctional drug delivery system for targeting tumor and its acidic microenvironment[J].J Control Release,2012,161:884-892.
[9]Cao DW,Chen JH,Feng M,et al.Nano-complexes from RGD-modified generation 1.0 polyamidoamine based copolymers used for intravascular gene release to prevent restenosis[J].Nanomedicine,2016,11:335-375.
[10]Zeng X,Pan SR,Li J,et al.A novel dendrimer based on poly(L-glutamic acid)derivatives as an efficient and biocompatible gene delivery vector[J].Nanotechnology,2011,22:375102.
[11]Cao DW,Chen JH,Pan SR,et al.Divalent folate modification on PEG:an effective strategy for improving the cellular uptake and targetability of PEGylated polyamidomine-polyethylenimine copolymer[J].Mol Pharm,2015,12:240-252.
[12]Kong SY,Tang GT,PEI YY,et al.PEGylated polyamidoamine dendrimer/methotrexate complex:pharmacokinetics and anti-tumor activity in normal and tumor-bearing rodents[J].Acta Pharm Sin(药学学报),2009,44:85-90.
[13]Lu JJ,Wu ZH,Ping QN.The effect of polyamidoamine(PAMAM)dendrimers on the solubility and pharmacokinetics of breviscapine[J].Acta Pharm Sin(药学学报),2009,44:197-202.
[14]DufèC,Uchegbu IF,Schatzlein AG.Dendrimers in gene delivery[J].Adv Drug Deliv Rev,2005,57:2177-2202.
[15]Wu HM,Pan SR,Chen MW,et al.A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection[J].Biomaterials,2011,32:1619-1634.
[16]Cao DW,Qin LH,Chen JH,et al.Transfection activity and the mechanism of PDNA-complexes based on the hybrid of low-generation PAMAM and branched PEI-1.8K[J].Mol Biosyst,2013,9:3175-3186.
[17]Wen YT,Guo ZH,Du Z,et al.Serum tolerance and endosomal escape capacity of histidine-modified p DNA-loaded complexes based on polyamidoamine dendrimer derivatives[J].Biomaterials,2012,33:8111-8121.
[18]Arima H,Yamashita S,Mori Y,et al.In vitro and in vivo gene delivery mediated by lactosylated dendrimer/a-cyclodextrin conjugates(G2)into hepatocytes[J].J Control Release,2010,146:106-117.
[19]Pan SR,Cao DW,Fang R,et al.Cellular uptake and transfection activity of DNA complexes based on poly(ethylene glycol)-poly(L-glutamine)copolymer with PAMAM G2[J].JMater Chem B,2013,1:5114-5127.
[20]Yang C,Li HZ,Goh SH,et al.Cationic star polymers consisting ofα-cyclodextrin core and oligoethylenimine arms as nonviral gene delivery vectors[J].Biomaterials,2007,28:3245-3254.
[21]Huang H,Cao DW,Qin LH,et al.Dilution-stable PAMAMG1-grafted polyrotaxane supermolecules deliver gene into cells through a caveolae-dependent pathway[J].Mol Pharm,2014,11:2323-2333.