Please wait a minute...
J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (6): 657-667    DOI: 10.3785/j.issn.1008-9292.2019.12.11
Synthesis and cell biological properties of polyaspartic acid drug/gene vector
SHEN Jie1,2(),WANG Qiwen3,GAO Dongruo1,LYU Yuanyuan1,TANG Guping2,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Chemistry, Zhejiang University, Hangzhou 310028, China
3. Department of Cardiovascular Medicine, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
Download: HTML( 3 )   PDF(7966KB)
Export: BibTeX | EndNote (RIS)      


Objective: Taking polysuccinimide as the main chain, amine side chain and alkyl side chain were grafted to prepare the drug/gene co-delivery vector. The property of the polymers with various side links were investigated to select an optimal vector. Methods: Poly-D, L-polysuccinimide was synthesized by polymerization reaction of D, L-aspartic acid as monomer. Therefore, N, N-dimethylenedipropyl-triamine and 3, 3'-diaminodipropylamine were grafted with dodecylamine/adecylamine/octadecylamine at different proportions by ring-opening reaction to obtain amphiphilic PEECs. The structure of the material was confirmed by 1H NMR; the particle size and surface potential of the micelles were measured by dynamic light scattering; the critical micelle concentration (CMC) was determined by pyrene fluorescent probe; the RNA blocking ability was characterized by agarose gel electrophoresis; the release behavior of the PEECs was examined and the cytotoxicity, cellular uptake and gene silencing efficiency of the PEECs were studied at the cellular level. Results: A series of PEECs with different grafting rates was successfully synthesized. The particle sizes and surface potential of the PEEC derived micelles were between 250 nm and 350 nm and 27 mV and 45 mV, respectively, with a small CMC value. The RNA binding ratio of PEECs was at a mass ratio of about 0.8:1. MTT assay demonstrated that PEEC micelles had certain cytotoxicity. PEECs had excellent micelle formation, drug-loading and gene binding abilities, particularly, PEEC16-2 showed high gene silencing efficiency at the cellular level. Conclusion: PEECs are able to co-delivery drug and gene, and PEEC16-2 micelles have the best ability of drug encapsulation and gene delivery.

Key wordsAspartic acid      Drug carriers      Genetic therapy      Nanoparticles      Neoplasms     
Received: 30 January 2019      Published: 19 January 2020
CLC:  R943  
Corresponding Authors: TANG Guping     E-mail:;
Cite this article:

SHEN Jie,WANG Qiwen,GAO Dongruo,LYU Yuanyuan,TANG Guping. Synthesis and cell biological properties of polyaspartic acid drug/gene vector. J Zhejiang Univ (Med Sci), 2019, 48(6): 657-667.

URL:     OR


目的: 以聚天冬氨酸作为主链,接枝胺基侧链和烷基侧链制备药物/基因共运输载体,考察不同侧链接枝率形成的纳米粒载体的载药及载基因性能,并筛选出性能较优的载体。方法: 以D,L-天冬氨酸为单体,通过聚合反应合成聚天冬氨酸,然后以不同比例的N,N-二甲基亚二丙基三胺、3,3'-二氨基二丙胺和十二胺/十六胺/十八胺进行开环接枝,得到两亲性聚天冬氨酸-3,3'-二氨基二丙胺-N,N-二甲基亚二丙基三胺-烷基胺(PEEC)材料。采用氢核磁共振确认PEEC结构,用动态光散射法测定纳米粒胶束的粒径和表面电位,用芘荧光探针法测定临界胶束浓度,用琼脂糖凝胶电泳表征RNA阻滞能力,考查载药胶束的释放行为并且在细胞层面上研究材料的细胞毒性、细胞摄取和基因沉默效率。结果: 成功合成了不同接枝率的PEEC材料,纳米粒胶束的粒径为250~350 nm,表面电位为27~45 mV,拥有较小的临界胶束浓度值,RNA阻滞的质量比约为0.8:1;PEEC胶束有一定的细胞毒性,其载药能力和基因绑定能力良好,其中PEEC16-2在细胞水平上表现出较高的基因沉默效率。结论: PEEC具有共载药物/基因的能力,其中PEEC16-2胶束具有良好的药物包载和基因运输能力。

关键词: 天冬氨酸,  药物载体,  基因治疗,  纳米粒子,  肿瘤 
样品 聚天冬氨酸(g) 烷基胺(g) N,N-二甲基亚二丙基三胺(mL) 3,3′-二氨基二丙胺(mL)
PEEC12-1 1.0 0.10 1.0 1.3
PEEC12-2 1.0 0.20 1.0 1.3
PEEC16-1 1.0 0.27 1.0 1.7
PEEC16-2 1.0 0.40 1.0 1.7
PEEC18-1 1.0 0.15 1.0 1.7
PEEC18-2 1.0 0.30 1.0 1.7
Tab 1 Raw materials for synthesis of polysuccinimide-dipropylenetriamine-N, N-dimethyldipropyiene-tramine (PEEC)
Fig 1 Synthesis of polysuccinimide-dipropylenetriamine-N, N-dimethyldipro-pyien-etramine (PEEC)
Fig 2 1H NMR spectra of polysuccinimide-dipropylenetriamine-N, N-dimethyldipropyienetramine (PEEC)
样品 x y z 烷基链接枝率(%) 粒径(nm) 表面电位(mV) 临界胶束浓度(μg/mL)
PEEC12-1 0.295 0.573 0.054 5.9 308±6 32.0±0.6 43.8
PEEC12-2 0.217 0.538 0.134 15.1 279±16 29.0±1.4 21.6
PEEC16-1 0.339 0.507 0.054 6.0 342±32 37.4±2.6 45.9
PEEC16-2 0.396 0.722 0.157 12.3 302±20 31.8±2.5 4.4
PEEC18-1 0.383 0.673 0.031 2.9 341±28 42.5±2.1 42.7
PEEC18-2 0.966 1.440 0.179 6.9 326±24 37.2±1.1 28.3
Tab 2 Grafting rate, particle size, surface potential and critical micelle concentration of polysuccinimide-dipropylenetriamine-N, N-dimethyldipropyienetramine (PEEC) ($\bar x \pm s$, n=3)
Fig 3 Critical micelle concentration of polysuccinimide-dipropylenetriamine-N, N-dimethyldipropyienetramine (PEEC)
Fig 4 RNA blocking ability of polysuccinimide-dipropylenetriamine-N, N-dimethyldipro-pyienetramine (PEEC) micelles (agarose gel electrophoresis)
Fig 5 Effect of polysuccinimide-dipropylene-triamine-N, N-dimethyldipropyienetramine (PEEC) micelles on cell viability
Fig 6 In vitro release curve of doxtaxol loaded PEEC16-2 micelles (n=3)
Fig 7 Cellular uptake of PEEC16-2/siRNA complex
Fig 8 Green fluorescent protein silencing results of PEEC16-2/siGFP (n=3)
Fig 9 Cellular uptake of PEEC16-2/DOX/siRNA
[1]   NAKAYAMA Y . Hyperbranched polymeric "star vectors" for effective DNA or siRNA delivery[J]. Acc Chem Res, 2012, 45 (7): 994- 1004
doi: 10.1021/ar200220t
[2]   BAO X , WANG W , WANG C et al. A chitosan-graft-PEI-candesartan conjugate for targeted co-delivery of drug and gene in anti-angiogenesis cancer therapy[J]. Biomaterials, 2014, 35 (29): 8450- 8466
doi: 10.1016/j.biomaterials.2014.06.025
[3]   CASETTARI L , VLLASALIU D , LAM J K et al. Biomedical applications of amino acid-modified chitosans:a review[J]. Biomaterials, 2012, 33 (30): 7565- 7583
doi: 10.1016/j.biomaterials.2012.06.104
[4]   SONG H Q , LI R Q , DUAN S et al. Ligand-functionalized degradable polyplexes formed by cationic poly(aspartic acid)-grafted chitosan-cyclodextrin conjugates[J]. Nanoscale, 2015, 7 (13): 5803- 5814
doi: 10.1039/C4NR07515C
[5]   ZHU H , DONG C , DONG H et al. Cleavable PEGylation and hydrophobic histidylation of polylysine for siRNA delivery and tumor gene therapy[J]. ACS Appl Mater Interfaces, 2014, 6 (13): 10393- 10407
doi: 10.1021/am501928p
[6]   KODAMA Y , NAKAMURA T , KUROSAKI T et al. Biodegradable nanoparticles composed of dendrigraft poly-L-lysine for gene delivery[J]. Eur J Pharm Biopharm, 2014, 87 (3): 472- 479
doi: 10.1016/j.ejpb.2014.04.013
[7]   LUO K , LI C , WANG G et al. Peptide dendrimers as efficient and biocompatible gene delivery vectors:Synthesis and in vitro characterization[J]. J Control Release, 2011, 155 (1): 77- 87
doi: 10.1016/j.jconrel.2010.10.006
[8]   MASTORAKOS P , KAMBHAMPATI S P , MISHRA M K et al. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells[J]. Nanoscale, 2015, 7 (9): 3845- 3856
doi: 10.1039/C4NR04284K
[9]   KUMAR A , YELLEPEDDI V K , VANGARA K K et al. Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues[J]. J Drug Target, 2011, 19 (9): 770- 780
doi: 10.3109/1061186X.2011.568061
[10]   WU H M , PAN S R , CHEN M W et al. A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection[J]. Biomaterials, 2011, 32 (6): 1619- 1634
doi: 10.1016/j.biomaterials.2010.09.045
[11]   GIAMMONA G , CAVALLARO G , FONTANA G et al. Coupling of the antiviral agent zidovudine to polyaspartamide and in vitro drug release studies[J]. J Control Release, 1998, 54 (3): 321- 331
doi: 10.1016/S0168-3659(98)00020-0
[12]   MIYATA K , OBA M , NAKANISHI M et al. Polyplexes from poly(aspartamide) bearing 1, 2-diaminoethane side chains induce pH-selective, endosomal membrane destabilization with amplified transfection and negligible cytotoxicity[J]. J Am Chem Soc, 2008, 130 (48): 16287- 16294
doi: 10.1021/ja804561g
[13]   CHENG H , LI Y Y , ZENG X et al. Protamine sulfate/poly(L-aspartic acid) polyionic complexes self-assembled via electrostatic attractions for combined delivery of drug and gene[J]. Biomaterials, 2009, 30 (6): 1246- 1253
doi: 10.1016/j.biomaterials.2008.11.002
[14]   WANG J L , TANG G P , SHEN J et al. A gene nanocomplex conjugated with monoclonal antibodies for targeted therapy of hepatocellular carcinoma[J]. Biomaterials, 2012, 33 (18): 4597- 4607
doi: 10.1016/j.biomaterials.2012.02.045
[15]   SHEN J , ZHAO D J , LI W et al. A polyethylenimine-mimetic biodegradable polycation with proper amine compositions for efficient gene delivery[J]. Biomaterials, 2013, 34 (18): 4520- 4531
doi: 10.1016/j.biomaterials.2013.02.068
[16]   赵丹军.聚天冬氨酸的衍生物作为基因载体的研究[D].杭州: 浙江大学, 2011.
ZHAO Danjun. Derivatives of poly(D, L-aspartic acid) used for gene delivery[D]. Hangzhou: Zhejiang University, 2011. (in Chinese)
[1] LIU Jingwen,YANG Xinglian,SHEN Kaili,ZENG Linghui,SUN Yan. Chloroxoquinoline inhibits invasion in breast cancer via down-regulating Rho/Rho kinase signaling pathway[J]. J Zhejiang Univ (Med Sci), 2019, 48(6): 631-637.
[2] CHEN Dianyu,QI Ming. Research progress on uniparental disomy in cancer[J]. J Zhejiang Univ (Med Sci), 2019, 48(5): 560-566.
[3] MA Panpan,CAI Lijun,LYU Bin,YUE Min. Application of probe-based confocal laser endomicroscopy in diagnosis of gastric carcinoma and precancerous lesions[J]. J Zhejiang Univ (Med Sci), 2019, 48(5): 504-510.
[4] HUANG Shumin,ZHAO Zhengyan. Advances in newborn screening and immune system reconstitution of severe combined immunodeficiency[J]. J Zhejiang Univ (Med Sci), 2019, 48(4): 351-357.
[5] ZHANG Dandan,WANG Junmei. Prenatal diagnosis and management of fetal hepatic hemangioma[J]. J Zhejiang Univ (Med Sci), 2019, 48(4): 439-445.
[6] WANG Qingmei, XU Qianzi, WEI Anyi, CHEN Shishuo, ZHANG Chong, ZENG Linghui. High dose vitamin C inhibits proliferation of breast cancer cells through reducing glycolysis and protein synthesis[J]. J Zhejiang Univ (Med Sci), 2019, 48(3): 296-302.
[7] ZHU Ziling, TAN Jing, DENG Hong. Nucleus translocation of membrane/cytoplasm proteins in tumor cells[J]. J Zhejiang Univ (Med Sci), 2019, 48(3): 318-325.
[8] ZHANG Yunzhu, ZHU Chunpeng, LU Xinliang. Advances in serum biomarkers for early diagnosis of gastric cancer[J]. J Zhejiang Univ (Med Sci), 2019, 48(3): 326-333.
[9] XU A'qiao,HE Hongqin,SHI Qiujun,LI Zhiqing,ZHANG Shengjian. Digital breast tomosynthesis in diagnosis of dense breast lesions[J]. J Zhejiang Univ (Med Sci), 2019, 48(2): 186-192.
[10] SHEN Xiameng,LYU Weiguo. Research advances on the role of exosomes in chemotherapy resistance of ovarian cancer[J]. J Zhejiang Univ (Med Sci), 2019, 48(1): 116-120.
[11] TANG Hexiao,BAI Yuquan,SHEN Wulin,ZHAO Jinping. Research progress on interleukin-6 in lung cancer[J]. J Zhejiang Univ (Med Sci), 2018, 47(6): 659-664.
[12] SHEN Hong,JI Feng. Efficacy and safety of digestive tract stent placement under direct visual endoscopy without X-ray monitoring in treatment of digestive tract stricture[J]. J Zhejiang Univ (Med Sci), 2018, 47(6): 643-650.
[13] LI Gaopeng,HE Jia,WANG Qingqing. Progress on cancer associated fibroblasts in tumor immunoregulation[J]. J Zhejiang Univ (Med Sci), 2018, 47(5): 558-563.
[14] WANG Xiaoling,OUYANG Xumei,SUN Xiaoyi. Application of mesenchymal stem cells in antineoplastic drugs delivery for tumor-targeted therapy[J]. J Zhejiang Univ (Med Sci), 2018, 47(5): 525-533.
[15] DI Chenhong,JIN Fan. Value of combined detection of claudin 4 and high-risk human papilloma virus in high-grade squamous intraepithelial lesion and cervix squamous cell carcinoma[J]. J Zhejiang Univ (Med Sci), 2018, 47(4): 344-350.