生物可裂解树状大分子组装体与智能型基因传递系统
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摘要
以精氨酸外围功能化、硫辛酸内核功能化的肽类树状大分子为组装基元,在亲疏水作用驱动下形成树状大分子组装体;并通过催化量二硫苏糖醇引发组装体内核的硫辛酸残基交联而获得纳米结构稳定、胞内氧化还原可裂解的智能型基因载体系统.双功能化肽类树状大分子具有精确可控的分子结构;在水溶液中组装、交联后,形成粒径约为72 nm、电位为+30.1 m V的树状大分子组装体.树状大分子组装体能够有效压缩和保护DNA,增强DNA对核酸酶的抵抗力,且具有氧化还原响应性、实现DNA的胞内定点可控释放.树状大分子组装体与DNA复合物(A/P=20)在有血清条件下对HEK 293、Hep G2和Hela 3种细胞系的转染效率均优于阳性对照组PEI(Mw=2.5×104,N/P=10,无血清)的转染效果.同时,该树状大分子组装体还具有良好的血清耐受性及生物相容性.
Development of supramolecular dendritic systems becomes a promising strategy on fabrication of efficient nanoplatforms for therapeutic agent delivery. Arginine-rich and lipoic acid-functionalized peptide dendrons were accurately synthesized using divergent approach and characterized with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Critical aggregation concentration of the amphiphilic peptide dendron was determined to be 209.8 μg/m L using pyrene as fluorescence probe. After supramolecular self-assembly of these peptide dendrons into nanoparticles in water, a catalytic amount of dithiothreitol(DTT) could induce polymerization to generate cross-linked and bio-cleavable dendritic systems(BCDSs) for improving the gene delivery efficiency in normal cells and tumor cells. The size of the disulfide cross-linked dendritic systems became much smaller( ~ 72 nm) than the size of the non-crosslinked dendritic assemblies( ~ 107 nm), which agreed well with the transmission electron microscope results. Agarose gel electrophoresis retardation assay indicated BCDSs completely condensed DNA at an A/P ratio of 5. An appropriate complex size of 80 nm was found at A/P of 20 with uniform spherical nanostructure. The BCDSs/DNA complex could efficiently protect DNA against DNase degradation, while reductive condition(10 mmol/L DTT) could induce the disintegration of nanocomplex and the release of DNA. Then, the confocal laser scan microscopy images indicated that intracellular reductive condition triggered cleavage of disulfide-stabilized supramolecular dendritic system and deliveried DNA into nucleus. Owing to the efficient DNA internalization and redox-responsive release of the BCDSs/DNA complexes(A/P = 20), green fluorescent protein expression was much better than the positive control of PEI group(N/P = 10, in the absence of fetal bovine serum). Quantitative result demonstrated that BCDSs could largely enhance the transfection efficiency of p GL3-Luc by 4.2 times, 2.7 times and 3.4 times in HEK 293, Hep G2 and Hela cell lines as compared with positive PEI group. Moreover, BCDSs were obviously nontoxic to all the three cell lines even at a high concentration of 60 μg/m L with more than 90% cell survived. However, PEI exhibited serious toxicity at low concentration of 10 μg/m L. This tailor-made molecular and supramolecular fabrication of biological cleavable dendritic systems holds great potentials in developing smart nanoplatforms for satisfactory gene delivery.
Development of supramolecular dendritic systems becomes a promising strategy on fabrication of efficient nanoplatforms for therapeutic agent delivery. Arginine-rich and lipoic acid-functionalized peptide dendrons were accurately synthesized using divergent approach and characterized with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Critical aggregation concentration of the amphiphilic peptide dendron was determined to be 209.8 μg/m L using pyrene as fluorescence probe. After supramolecular self-assembly of these peptide dendrons into nanoparticles in water, a catalytic amount of dithiothreitol(DTT) could induce polymerization to generate cross-linked and bio-cleavable dendritic systems(BCDSs) for improving the gene delivery efficiency in normal cells and tumor cells. The size of the disulfide cross-linked dendritic systems became much smaller( ~ 72 nm) than the size of the non-crosslinked dendritic assemblies( ~ 107 nm), which agreed well with the transmission electron microscope results. Agarose gel electrophoresis retardation assay indicated BCDSs completely condensed DNA at an A/P ratio of 5. An appropriate complex size of 80 nm was found at A/P of 20 with uniform spherical nanostructure. The BCDSs/DNA complex could efficiently protect DNA against DNase degradation, while reductive condition(10 mmol/L DTT) could induce the disintegration of nanocomplex and the release of DNA. Then, the confocal laser scan microscopy images indicated that intracellular reductive condition triggered cleavage of disulfide-stabilized supramolecular dendritic system and deliveried DNA into nucleus. Owing to the efficient DNA internalization and redox-responsive release of the BCDSs/DNA complexes(A/P = 20), green fluorescent protein expression was much better than the positive control of PEI group(N/P = 10, in the absence of fetal bovine serum). Quantitative result demonstrated that BCDSs could largely enhance the transfection efficiency of p GL3-Luc by 4.2 times, 2.7 times and 3.4 times in HEK 293, Hep G2 and Hela cell lines as compared with positive PEI group. Moreover, BCDSs were obviously nontoxic to all the three cell lines even at a high concentration of 60 μg/m L with more than 90% cell survived. However, PEI exhibited serious toxicity at low concentration of 10 μg/m L. This tailor-made molecular and supramolecular fabrication of biological cleavable dendritic systems holds great potentials in developing smart nanoplatforms for satisfactory gene delivery.
引文
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24 Xu Xianghui(徐翔晖),Li Caixia(李彩侠),Li Haiping(李海平),Liu Rong(刘荣),Jiang Chao(江超),Wu Yao(吴尧),He Bin(何斌),Gu Zhongwei(顾忠伟).Scientia Sinica Chimica(中国科学?化学),2011,(2):359?367
25 Luo X,Huang F,Qin S,Wang H,Feng J,Zhang X,Zhuo R.Biomaterials,2011,32(36):9925?9939
2 Gu Zhongwei(顾忠伟),Luo Kui(罗奎),She Wenchuan(佘汶川),Wu Yao(吴尧),He Bin(何斌).Scientia Sinica Chimica(中国科学?化学),2010,40(3):210?236
3 Leiro V,Garcia J o P,Tomás H,Pêgo A P.Bioconjugate Chem,2015,26(7):1182?1197
4 Menjoge A R,Kannan R M,Tomalia D A.Drug Discov Today,2010,15(5):171?185
5 Hawker C J,Wooley K L.Science,2005,309(5738):1200?1205
6 Walter M V,Malkoch M.Chem Soc Rev,2012,41(13):4593–4609
7 Xu X,Yuan H,Chang J,He B,Gu Z.Angew Chem Int Ed,2012,51(13):3130?3133
8 Xu X,Li Y,Li H,Liu R,Sheng M,He B,Gu Z.Small,2014,10(6):1133?1140
9 Li Y,Lai Y,Xu X,Zhang X,Wu Y,Hu C,Gu Z.Nanomedicine NBM,2016,12(2):355?364
10 Zhang Z,Zhang X,Xu X,Li Y,Li Y,Zhong D,He Y,Gu Z.Adv Funct Mater,2015,25(33):5250?5260
11 Xu X,Jian Y,Li Y,Zhang X,Tu Z,Gu Z.ACS Nano,2014,8(9):9255?9264
12 Li Y,Li Y,Zhang X,Xu X,Zhang Z,Hu C,He Y,Gu Z.Theranostics,2016,6(9):1293?1305
13 Li S,Huang L.Gene Ther,2006,13(18):1313?1319
14 Guo X,Huang L.Acc Chem Res,2011,45(7):971?979
15 Giacca M,Zacchigna S.J Controll Release,2012,161(2):377?388
16 Tian H,Chen J,Chen X.Small,2013,9(12):2034?2044
17 Jin L,Zeng X,Liu M,Deng Y,He N.Theranostics,2014,4(3):240?255
18 Xu Xianghui(徐翔晖),Li Yunkun(李芸焜),Jian Yeting(简也挺),Wang Gang(王刚),He Bin(何斌),Gu Zhongwei(顾忠伟).Acta Polymerica Sinica(高分子学报),2012,(10):1128?1135
19 Xu X,Jiang Q,Zhang X,Nie Y,Zhang Z,Li Y,Cheng G,Gu Z.J Mater Chem B,2015,3(35):7006?7010
20 Son S,Namgung R,Kim J,Singha K,Kim W J.Acc Chem Res,2011,45(7):1100?1112
21 Shim M S,Kwon Y J.Adv Drug Delivery Rev,2012,64(11):1046?1059
22 Zhang X,Zhang Z,Xu X,Li Y,Li Y,Jian Y,Gu Z.Angew Chem Int Ed,2015,54(14):4289?4294
23 Li H,Xu X,Li Y,Geng Y,He B,Gu Z.Polym Chem,2013,4(7):2235?2238
24 Xu Xianghui(徐翔晖),Li Caixia(李彩侠),Li Haiping(李海平),Liu Rong(刘荣),Jiang Chao(江超),Wu Yao(吴尧),He Bin(何斌),Gu Zhongwei(顾忠伟).Scientia Sinica Chimica(中国科学?化学),2011,(2):359?367
25 Luo X,Huang F,Qin S,Wang H,Feng J,Zhang X,Zhuo R.Biomaterials,2011,32(36):9925?9939