基于磷酸酯的高分子作为药物及基因输运载体的研究
|
摘要
研究和开发具有良好性能的药物和基因输送载体是目前生物医学领域的重要研究方向。本论文针对药物和基因的输送设计合成了两种性能优越的载体材料,并对它们的性能进行了研究。
我们设计合成了一种还原响应性的纳米药物载体,载体材料是由二硫键连接的两亲性嵌段聚合物PCL-SS-PEEP,含有疏水性聚己内酯嵌段和亲水性聚2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷嵌段。载体材料的化学结构经凝胶渗透色谱(GPC)和1H NMR等表征手段验证。PCL-SS-PEEP在水溶液中自组装形成胶束纳米粒,芘荧光探针法测得其临界胶束浓度为3.1×10-3 mg mL-1,透射电子显微镜(TEM)观察和动态光散射(DLS)检测表明胶束的粒径在100 nm左右且分布均匀。该胶束与10 mM谷胱甘肽(GSH)共培养时,DLS检测颗粒粒径随时间延长显著增大,显示在GSH作用下载体聚合物二硫键断裂破坏了胶束的结构,并因此造成团聚。通过疏水相互作用可以将阿霉素(DOX)包载到胶束内核中,而且DOX从胶束的释放具有GSH浓度响应性,在较高GSH浓度下DOX的释放更快。在细胞实验中,用谷胱甘肽单乙酯(GSH-OEt)处理A549细胞,构建了胞内富集GSH的细胞模型,将载有DOX的胶束纳米粒和A549细胞培养,激光共聚焦和流式细胞检测表明,经GSH-OEt处理的A549细胞内阿霉素荧光比未经GSH-OEt处理的A549细胞增强,表明GSH-OEt处理的A549细胞内高的GSH浓度造成更多的DOX在胞内快速释放。此外,在相同DOX剂量下,和载有DOX的胶束纳米粒共培养的细胞存活率随加入GSH-OEt浓度的增加而下降,也说明PCL-SS-PEEP胶束的GSH响应性的药物释放行为。这种智能化的胞内药物输送和释放为药物输送的纳米载体的设计提供了新的思路。
在基因输送载体的研究中,通过聚乙烯亚胺和2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷的化学反应获得了具有不同磷酸酯修饰度的聚乙烯亚胺衍生物(PEI-EEP),利用1H和31P NMR表征了聚合物的结构并初步推测了反应进行的机理。这一系列PEI-EEP聚合物表现出与PEI类似的缓冲能力,在较低的N/P比下完全结合DNA。我们用DLS的手段研究了不同修饰度的PEI-EEP与DNA在不同N/P下结合形成复合物的粒径和电势的变化趋势,同时用噻唑蓝(MTT)细胞活性证明PEI-EEP聚合物与PEI相比,对HEK293和HeLa细胞均显示低的细胞毒性,而且随修饰度的增加而下降。PEI-PEEP对HEK293和HeLa的体外转染实验结果表明其转染效率与修饰度密切相关,每个PEI分子中含EEP修饰度为52和75的PEI-EEP聚合物表现出比PEI高的转染效率,当EEP修饰度增加至116个时,聚合物的转染效率则显著下降。此外,与无血清时相比,PEI-EEP聚合物在血清存在时表现出更高的转染效率。综合上述,PEI-EEP聚合物可以同时表现出高的转染效率和低的细胞毒性,表明PEI-EEP有潜力成为基因输送的载体材料。
Development of drug and gene delivery carriers with fine properties is a hot topic of biomedicine research. In the thesis, two polymers with unique properties were designed and synthesized as the carriers of drug and gene delivery systems, respectively. The structures and properties of the two polymers were well characterized.
A novel glutathione-responsive amphiphilic block copolymer consisting of polycaprolactone and polyphosphoester with intervening disulfide bond, termed as PCL-SS-PEEP, was synthesized. The block copolymer was well characterized by 1H NMR and gel permeation chromatograph. The amphiphilic copolymer formed micelles in aqueous solution and the critical micelle concentration was 3.1×10-3 mg mL-1. Transmition electron microscopy and dynamic light scattering (DLS) measurements demonstrated that the micelles were relatively uniform in size with the average diameter ca. 100 nm. The micelles displayed glutathione-dependent size changes according to DLS measurement. Doxorubicin [1] was encapsulated in the micelles and its in vitro release profile demonstrated that more DOX was released at a faster rate in the presence of glutathione (GSH). At cellular level, the cells were pretreated with glutathione monoester (GSH-OEt) at different concentrations to manipulate the intracellular GSH concentration. The confocal laser scanning microscopic and flow cytometric analyses indicated that more intensive fluorescence of DOX were detected in cells pretreated by GSH-OEt due to the GSH-triggered DOX release. The DOX-loaded micelles also exhibited higher cytotoxicity in GSH-OEt-pretreated A549 cells. This indicated that the GSH-responsive PCL-SS-PEEP micelles are promising for intracellular drug delivery and it also provides a new paradigm for designing drug delivery vehicles.
Cyclic phosphate monomer ethyl ethylene phosphate (EEP) modified poly(ethylenimine) (PEI), denoted as PEI-EEP, was synthesized for gene delivery. Three PEI-EEP polymers were synthesized and their structures were characterized by 1H and 31P NMR methods. The buffering capabilities of PEI-EEPs were examined by acid–base titration and the titration profiles showed that all of the PEI-EEPs had the similar buffering capability as unmodified PEI. Physiochemical characteristics of PEI-EEP/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and zeta potential measurements. All of the PEI-EEPs were able to condense DNA efficiently at N/P ratios higher than 0.5:1. The particle sizes of PEI-EEPs/DNA complexes were around 160–250 nm, while the surface charges were around 30-50 mV at the N/P ratios ranging from 10:1 to 50:1. In vitro cell viability and transfection ability were evaluated in HEK293 and HeLa cells using PEI as the control. The cytotoxicity of PEI-EEPs and PEI-EEPs/DNA complexes were lower than that of PEI and its complexes with DNA. The in vitro transfection experiments indicated that the transfection efficiency of PEI-EEP/DNA complexes were related to the modification degrees by the phosphate. When the phosphate units per PEI were 52 and 75, the PEI-EEP/DNA complexes exhibited comparable or even higher transfection ability than PEI/DNA complex did at its optimal N/P ratio of 10:1 in the absence of serum. However, when the phosphate units increased to 116 per PEI, the transfection efficiency dramatically reduced.
我们设计合成了一种还原响应性的纳米药物载体,载体材料是由二硫键连接的两亲性嵌段聚合物PCL-SS-PEEP,含有疏水性聚己内酯嵌段和亲水性聚2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷嵌段。载体材料的化学结构经凝胶渗透色谱(GPC)和1H NMR等表征手段验证。PCL-SS-PEEP在水溶液中自组装形成胶束纳米粒,芘荧光探针法测得其临界胶束浓度为3.1×10-3 mg mL-1,透射电子显微镜(TEM)观察和动态光散射(DLS)检测表明胶束的粒径在100 nm左右且分布均匀。该胶束与10 mM谷胱甘肽(GSH)共培养时,DLS检测颗粒粒径随时间延长显著增大,显示在GSH作用下载体聚合物二硫键断裂破坏了胶束的结构,并因此造成团聚。通过疏水相互作用可以将阿霉素(DOX)包载到胶束内核中,而且DOX从胶束的释放具有GSH浓度响应性,在较高GSH浓度下DOX的释放更快。在细胞实验中,用谷胱甘肽单乙酯(GSH-OEt)处理A549细胞,构建了胞内富集GSH的细胞模型,将载有DOX的胶束纳米粒和A549细胞培养,激光共聚焦和流式细胞检测表明,经GSH-OEt处理的A549细胞内阿霉素荧光比未经GSH-OEt处理的A549细胞增强,表明GSH-OEt处理的A549细胞内高的GSH浓度造成更多的DOX在胞内快速释放。此外,在相同DOX剂量下,和载有DOX的胶束纳米粒共培养的细胞存活率随加入GSH-OEt浓度的增加而下降,也说明PCL-SS-PEEP胶束的GSH响应性的药物释放行为。这种智能化的胞内药物输送和释放为药物输送的纳米载体的设计提供了新的思路。
在基因输送载体的研究中,通过聚乙烯亚胺和2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷的化学反应获得了具有不同磷酸酯修饰度的聚乙烯亚胺衍生物(PEI-EEP),利用1H和31P NMR表征了聚合物的结构并初步推测了反应进行的机理。这一系列PEI-EEP聚合物表现出与PEI类似的缓冲能力,在较低的N/P比下完全结合DNA。我们用DLS的手段研究了不同修饰度的PEI-EEP与DNA在不同N/P下结合形成复合物的粒径和电势的变化趋势,同时用噻唑蓝(MTT)细胞活性证明PEI-EEP聚合物与PEI相比,对HEK293和HeLa细胞均显示低的细胞毒性,而且随修饰度的增加而下降。PEI-PEEP对HEK293和HeLa的体外转染实验结果表明其转染效率与修饰度密切相关,每个PEI分子中含EEP修饰度为52和75的PEI-EEP聚合物表现出比PEI高的转染效率,当EEP修饰度增加至116个时,聚合物的转染效率则显著下降。此外,与无血清时相比,PEI-EEP聚合物在血清存在时表现出更高的转染效率。综合上述,PEI-EEP聚合物可以同时表现出高的转染效率和低的细胞毒性,表明PEI-EEP有潜力成为基因输送的载体材料。
Development of drug and gene delivery carriers with fine properties is a hot topic of biomedicine research. In the thesis, two polymers with unique properties were designed and synthesized as the carriers of drug and gene delivery systems, respectively. The structures and properties of the two polymers were well characterized.
A novel glutathione-responsive amphiphilic block copolymer consisting of polycaprolactone and polyphosphoester with intervening disulfide bond, termed as PCL-SS-PEEP, was synthesized. The block copolymer was well characterized by 1H NMR and gel permeation chromatograph. The amphiphilic copolymer formed micelles in aqueous solution and the critical micelle concentration was 3.1×10-3 mg mL-1. Transmition electron microscopy and dynamic light scattering (DLS) measurements demonstrated that the micelles were relatively uniform in size with the average diameter ca. 100 nm. The micelles displayed glutathione-dependent size changes according to DLS measurement. Doxorubicin [1] was encapsulated in the micelles and its in vitro release profile demonstrated that more DOX was released at a faster rate in the presence of glutathione (GSH). At cellular level, the cells were pretreated with glutathione monoester (GSH-OEt) at different concentrations to manipulate the intracellular GSH concentration. The confocal laser scanning microscopic and flow cytometric analyses indicated that more intensive fluorescence of DOX were detected in cells pretreated by GSH-OEt due to the GSH-triggered DOX release. The DOX-loaded micelles also exhibited higher cytotoxicity in GSH-OEt-pretreated A549 cells. This indicated that the GSH-responsive PCL-SS-PEEP micelles are promising for intracellular drug delivery and it also provides a new paradigm for designing drug delivery vehicles.
Cyclic phosphate monomer ethyl ethylene phosphate (EEP) modified poly(ethylenimine) (PEI), denoted as PEI-EEP, was synthesized for gene delivery. Three PEI-EEP polymers were synthesized and their structures were characterized by 1H and 31P NMR methods. The buffering capabilities of PEI-EEPs were examined by acid–base titration and the titration profiles showed that all of the PEI-EEPs had the similar buffering capability as unmodified PEI. Physiochemical characteristics of PEI-EEP/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and zeta potential measurements. All of the PEI-EEPs were able to condense DNA efficiently at N/P ratios higher than 0.5:1. The particle sizes of PEI-EEPs/DNA complexes were around 160–250 nm, while the surface charges were around 30-50 mV at the N/P ratios ranging from 10:1 to 50:1. In vitro cell viability and transfection ability were evaluated in HEK293 and HeLa cells using PEI as the control. The cytotoxicity of PEI-EEPs and PEI-EEPs/DNA complexes were lower than that of PEI and its complexes with DNA. The in vitro transfection experiments indicated that the transfection efficiency of PEI-EEP/DNA complexes were related to the modification degrees by the phosphate. When the phosphate units per PEI were 52 and 75, the PEI-EEP/DNA complexes exhibited comparable or even higher transfection ability than PEI/DNA complex did at its optimal N/P ratio of 10:1 in the absence of serum. However, when the phosphate units increased to 116 per PEI, the transfection efficiency dramatically reduced.
引文
1. Ishida T, Kirchmeier MJ, Moase EH, Zalipsky S, Allen TM. 2001.Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells[J]. Biochimica Et Biophysica Acta-Biomembranes, 1515(2):144-158.
2. Harada A, Kataoka K. 2006. Supramolecular assemblies of block copolymers in aqueous media as nanocontainers relevant to biological applications[J]. Progress in Polymer Science, 31(11):949-982.
3. Majumdar S, Duvvuri S, Mitra AK. 2004.Membrane transporter/receptor-targeted prodrug design: strategies for human and veterinary drug development[J].Advanced Drug Delivery Reviews, 56(10):1437-1452.
4. Stella VJ, Nti-Addae KW. 2007. Prodrug strategies to overcome poor water solubility[J]. Advanced Drug Delivery Reviews, 59(7):677-694.
5. Chari RVJ. 1998. Targeted delivery of chemotherapeutics: tumor-activated prodrug therapy[J]. Advanced Drug Delivery Reviews, 31(1-2):89-104.
6. Wang YC, Tang LY, Sun TM, Li CH, Xiong MH, Wang J. 2008 .Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(epsilon-caprolactone) as drug carriers[J]. Biomacromolecules, 9(1):388-395.
7. De Smedt SC, Demeester J, Hennink WE. 2000.Cationic polymer based gene delivery systems[J]. Pharmaceutical Research ,17(2):113-126.
8. Wong SY, Pelet JM, Putnam D. 2007. Polymer systems for gene delivery-past, present, and future[J]. Progress in Polymer Science, 32(8-9):799-837.
9. Kircheis R, Wightman L, Robitza B, Ogris M, Brunner S, Wagner E. 2000. Polymer-based systems for tumor-targeted gene delivery[J]. Cancer Gene Therapy, 7(8):1206-1206.
10. Akinc A, Thomas M, Klibanov AM, Langer R. 2005. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis[J]. Journal of Gene Medicine, 7(5):657-663.
11. Lu L, Zhu X, Valenzuela RG, Currier BL, Yaszemski MJ. 2001. Biodegradable polymer scaffolds for cartilage tissue engineering[J]. Clinical Orthopaedics and Related Research, (391 Suppl):S251-270.
12. Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. 1998. Polymer concepts in tissue engineering[J]. Journal of Biomedical Materials Research,43(4):422-427.
13. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. 2006. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering[J]. Biomaterials, 27(18):3413-3431.
14. Seunarine K, Gadegaard N, Tormen M, Meredith DO, Riehle MO, Wilkinson CD.2006. 3D polymer scaffolds for tissue engineering[J]. Nanomed, 1(3):281-296.
15. Kallela I, Tulamo RM, Hietanen J, Pohjonen T, Suuronen R, Lindqvist C. 1999. Fixation of mandibular body osteotomies using biodegradable amorphous self-reinforced (70L : 30DL) polylactide or metal lag screws: an experimental study in sheep[J]. Journal of Cranio-Maxillofacial Surgery, 27(2):124-133.
16. Saikku-Backstrom A, Tulamo RM, Pohjonen T, Tormala P, Raiha JE, Rokkanen P. 1999. Material properties of absorbable self-reinforced fibrillated poly-96L/4D-lactide (SR-PLA96) rods; a study in vitro and in vivo[J]. Journal of Materials Science-Materials in Medicine, 10(1):1-8.
17. Savic R, Luo LB, Eisenberg A, Maysinger D. 2003. Micellar nanocontainers distribute to defined cytoplasmic organelles[J]. Science, 300(5619):615-618.
18. Torchilin VP. 2007. Micellar nanocarriers: pharmaceutical perspectives[J]. Pharmaceutical Research, 24(1):1-16.
19. Lee ES, Na K, Bae YH. 2005. Super pH-sensitive multifunctional polymeric micelle[J]. Nano Letters, 5(2):325-329.
20. Lee ES, Gao ZG, Kim D, Park K, Kwon IC, Bae YH. 2008. Super pH-sensitive multifunctional polymeric micelle for tumor pH(e) specific TAT exposure and multidrug resistance[J]. Journal of Controlled Release, 129(3):228-236.
21. Hoffman AS. 2000. Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations[J]. Clinical Chemistry, 46(9):1478-1486.
22. Gil ES, Hudson SA. 2004.Stimuli-reponsive polymers and their bioconjugates[J]. Progress in Polymer Science, 29(12):1173-1222.
23. Rijcken CJF, Soga O, Hennink WE, van Nostrum CF. 2007. Triggered destabilisation of polymeric micelles and vesicles by changing polymers polarity: An attractive tool for drug delivery[J]. Journal of Controlled Release, 120(3):131-148.
24. Liu SQ, Tong YW, Yang YY. 2005. Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) with varying compositions[J]. Biomaterials, 26(24):5064-5074.
25. Lin C, Zhong ZY, Lok MC, et al. 2007. Novel bioreducible poly(amido amine)s for highly efficient gene delivery[J]. Bioconjugate Chemistry, 18(1):138-145.
26. Lin C, Zhong ZY, Lok MC, Jiang XL, Hennink WE, Jan FJ, et al. 2006. Linear poly(amido amine)s with secondary and tertiary amino groups and variable amounts of disulfide linkages: Synthesis and in vitro gene transfer properties[J]. Journal of Controlled Release,28;116(2):130-137.
27. Lin C, Blaauboer CJ, Timoneda MM, et al. 2008 .Bioreducible poly(amido amine)s with oligoamine side chains: synthesis, characterization, and structural effects on gene delivery[J]. Journal of Controlled Release, 3;126(2):166-174.
28. Christensen LV, Chang CW, Kim WJ, et al. 2006. Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery[J]. Bioconjuguate Chemistry, 17(5):1233-1240.
29. Matsumoto S, Christie RJ, Nishiyama N et al. 2009. Environment-Responsive Block Copolymer Micelles with a Disulfide Cross-Linked Core for Enhanced siRNA Delivery[J]. Biomacromolecules,10(1):119-127.
30. Saito G, Swanson JA, Lee KD.2003.Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities[J]. Advanced Drug Delivery Review, 55(2):199-215.
31. Koo AN, Lee HJ, Kim SE, Chang JH, Park C, Kim C, et al. 2008. Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-mediated intracellular drug delivery[J]. Chemical Communications (Camb), Dec 28(48):6570-6572.
32. Xiao CS, Wang YC, Du JZ, Chen XS, Wang J.2006.Kinetics and mechanism of 2-ethoxy-2-oxo-1,3,2-dioxaphospholane polymerization initiated by stannous octoate[J]. Macromolecules, 39(20):6825-6831.
33. Ghosh S, Basu S, Thayumanavan S. 2006. Simultaneous and reversible functionalization of copolymers for biological applications[J]. Macromolecules, 39(17):5595-5597.
34. Carrot G, Hilborn JG, Trollsas M, Hedrick JL. 1999 .Two general methods for the synthesis of thiol-functional polycaprolactones[J]. Macromolecules, 32(16):5264-5269.
35. Du JZ, Chen DP, Wang YC, Xiao CS, Lu YJ, Wang J, et al. 2006. Synthesis and micellization of amphiphilic brush-coil block copolymer based on poly(epsilon-caprolactone) and PEGylated polyphosphoester[J]. Biomacromolecules, 7(6):1898-1903.
36. Li SD, Huang L. 2008. Pharmacokinetics and biodistribution of nanoparticles[J]. Molecular Pharmaceutics, 5(4):496-504.
37. Bae Y, Fukushima S, Harada A, Kataoka K.2003.Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change[J]. Angewandte Chemie International Edition, 42(38):4640-4643.
38. Thierry AR, Rabinovich P, Peng B, Mahan LC, Bryant JL, Gallo RC. 1997. Characterization of liposome-mediated gene delivery: Expression, stability and pharmacokinetics of plasmid DNA[J]. Gene Therapy, 4(3):226-237.
39. Sun X, Hai L, Wu Y, Hu HY, Zhang ZR. 2005. Targeted gene delivery to hepatoma cellsusing galactosylated liposome-polycation-DNA complexes (LPD)[J].Journal of Drug Targeting, 13(2):121-128.
40. Park JG, Moon IJ. 2003. Developments of DNA delivery system using the triple complex of DNA: Peptide: Liposome[J]. Molecular Therapy, 7(5):S217-S217.
41. Boulikas T. 1996. Liposome DNA delivery and uptake by cells (review) [J].Oncology Reports, 3(6):989-995.
42. Scaria P, Woodle MC. 2002. Polymer conjugates for gene delivery[M]. Abstracts of Papers of the American Chemical Society, 18;224:U471-U471.
43. Neu M, Germershaus O, Behe M, Kissel T. 2007. Bioreversibly crosslinked polyplexes of PEI and high molecular weight PEG show extended circulation times in vivo[J].Journal of Controlled Release, 124(1-2):69-80.
44. Ahn HH, Lee MS, Cho MH, Shin YN, Lee JH, Kim KS, et al. 2008. DNA/PEI nano-particles for gene delivery of rat bone marrow stem cells[J]. Colloids and Surfaces a-Physicochemical and Engineering Aspects, 313:116-120.
45. Nam YS, Kang HS, Park JY, Park TG, Han SH, Chang IS. 2003. New micelle-like polymer aggregates made from PEI-PLGA diblock copolymers: micellar characteristics and cellular uptake[J]. Biomaterials,24(12):2053-2059.
46. Wang DA, Narang AS, Kotb M, Gaber AO, Miller DD, Kim SW, et al. 2002. Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery[J]. Biomacromolecules, 3(6):1197-1207.
47. Godbey WT, Wu KK, Mikos AG. 1999.Poly(ethylenimine) and its role in gene delivery[J]. Journal of Controlled Release, 60(2-3):149-160.
48. Santos JL, Oramas E, Pego AP, Granja PL, Tomas H. 2009. Osteogenic differentiation of mesenchymal stem cells using PAMAM dendrimers as gene delivery vectors[J]. Journal of Controlled Release, 134(2):141-148.
49. Kim T, Seo HJ, Choi JS, Jang HS, Baek J, Kim K, et al. 2004. PAMAM-PEG-PAMAM: Novel triblock copolymer as a biocompatible and efficient gene delivery carrier[J]. Biomacromolecules, 5(6):2487-2492.
50. Lee JH, Lim YB, Choi JS, Lee Y, Kim TI, Kim HJ, et al. 2003. Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency[J]. Bioconjugate Chemistry, 14(6):1214-1221.
51. Erbacher P, Zou SM, Bettinger T, Steffan AM, Remy JS.1998. Chitosan-based vector/DNA complexes for gene delivery: Biophysical characteristics and transfection ability[J]. Pharmaceutical Research, 15(9):1332-1339.
52. Lee KY, Kwon IC, Kim YH, Jo WH, Jeong SY. 1998. Preparation of chitosan self-aggregates as a gene delivery system[J]. Journal of Controlled Release, 51(2-3):213-220.
53. Kim TH, Jiang HL, Nah JW, Cho MH, Akaike T, Cho CS. 2007. Receptor-mediated gene delivery using chitosan derivatives in vitro and in vivo[J].Macromolecular Symposia,249:137-144
54. Strand SP, Issa MM, Christensen BE, Varum KM, Artursson P. 2008.Tailoring of Chitosans for Gene Delivery: Novel Self-Branched Glycosylated Chitosan Oligomers with Improved Functional Properties[J]. Biomacromolecules, 9(11):3268-3276.
55. Mintzer MA, Simanek EE. 2009. Nonviral Vectors for Gene Delivery[J].Chemical Reviews, 109(2):259-302.
56. Forrest ML, Meister GE, Koerber JT, Pack DW. 2004. Partial acetylation of polyethylenimine enhances in vitro gene delivery[J]. Pharmaceutical Research, 21(2):365-371.
57. Rhaese S, von Briesen H, Rubsamen-Waigmann H, Kreuter J, Langer K. 2003. Human serum albumin-polyethylenimine nanoparticles for gene delivery[J].Journal of Controlled Release, 92(1-2):199-208.
58. Carrabino S, Di Gioia S, Copreni E, Conese M. 2005. Serum albumin enhances polyethylenimine-mediated gene delivery to human respiratory epithelial cells[J]. Journal of Gene Medicine, 7(12):1555-1564.
59. Gosselin MA, Guo WJ, Lee RJ. 2001. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine[J]. Bioconjugate Chemistry, 12(6):989-994.
60. Peng Q, Zhong Z, Zhuo R.2008. Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors[J].Bioconjuguate Chemistry,19(2):499-506.
61. Lu B, Xu XD, Zhang XZ, Cheng SX, Zhuo RX. 2008. Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies[J]. Biomacromolecules,9(10):2594-2600.
62. Xiong MP, Forrest ML, Ton G, Zhao A, Davies NM, Kwon GS.2007.Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery[J]. Biomaterials,28(32):4889-4900.
63. Sun YX, Xiao W, Cheng SX, Zhang XZ, Zhuo RX. 2008. Synthesis of (Dex-HMDI)-g-PEIs as effective and low cytotoxic nonviral gene vectors[J]. Journal of Controlled Release, 4;128(2):171-178.
64. Thomas M, Klibanov AM. 2002. Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 99(23):14640-14645.
65. Doody AM, Korley JN, Dang KP, Zawaneh PN, Putnam D. 2006. Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro[J]. Journal of Controlled Release, 116(2):227-237.
66. Zintchenko A, Philipp A, Dehshahri A, Wagner E. 2008. Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity[J].Bioconjugate Chemistry, 19(7):1448-1455.
67. Sun TM, Du JZ, Yan LF, Mao HQ, Wang J. 2008. Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery[J]. Biomaterials, 29(32):4348-4355.
68. Wang YC, Tang LY, Li Y, Wang J.2009.Thermoresponsive Block Copolymers of Poly(ethylene glycol) and Polyphosphoester: Thermo-Induced Self-Assembly, Biocompatibility, and Hydrolytic Degradation[J]. Biomacromolecules, 10(1):66-73.
69. Ashkenazi N, Zade SS, Segall Y, Karton Y, Bendikov M. 2005. Selective site controlled nucleophilic attacks in 5-membered ring phosphate esters: unusual C-O vs. common P-O bond cleavage[J]. Chemical Communications, (47):5879-5881.
70. Jiang X, Dai H, Ke CY, Mo X, Torbenson MS, Li Z, et al. 2007. PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion[J]. Journal of Control Release, 122(3):297-304.
71. Xu DM, Yao SD, Liu YB, Sheng KL, Hong J, Gong PJ, et al. 2007. Size-dependent properties of M-PEIs nanogels for gene delivery in cancer cells[J]. International Journal of Pharmaceutics, 338(1-2):291-296.
72. Tian HY, Xiong W, Wei JZ, Wang Y, Chen XS, Jing XB, et al. 2007. Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG[J]. Biomaterials,28(18):2899-2907.
73. Kunath K, von Harpe A, Fischer D, Peterson H, Bickel U, Voigt K, et al. 2003. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine[J]. Journal of Controlled Releas, 14;89(1):113-125.
74. Jiang X, Lok MC, Hennink WE. 2007. Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery[J]. Bioconjugate Chemistry, 18(6):2077-2084.
2. Harada A, Kataoka K. 2006. Supramolecular assemblies of block copolymers in aqueous media as nanocontainers relevant to biological applications[J]. Progress in Polymer Science, 31(11):949-982.
3. Majumdar S, Duvvuri S, Mitra AK. 2004.Membrane transporter/receptor-targeted prodrug design: strategies for human and veterinary drug development[J].Advanced Drug Delivery Reviews, 56(10):1437-1452.
4. Stella VJ, Nti-Addae KW. 2007. Prodrug strategies to overcome poor water solubility[J]. Advanced Drug Delivery Reviews, 59(7):677-694.
5. Chari RVJ. 1998. Targeted delivery of chemotherapeutics: tumor-activated prodrug therapy[J]. Advanced Drug Delivery Reviews, 31(1-2):89-104.
6. Wang YC, Tang LY, Sun TM, Li CH, Xiong MH, Wang J. 2008 .Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(epsilon-caprolactone) as drug carriers[J]. Biomacromolecules, 9(1):388-395.
7. De Smedt SC, Demeester J, Hennink WE. 2000.Cationic polymer based gene delivery systems[J]. Pharmaceutical Research ,17(2):113-126.
8. Wong SY, Pelet JM, Putnam D. 2007. Polymer systems for gene delivery-past, present, and future[J]. Progress in Polymer Science, 32(8-9):799-837.
9. Kircheis R, Wightman L, Robitza B, Ogris M, Brunner S, Wagner E. 2000. Polymer-based systems for tumor-targeted gene delivery[J]. Cancer Gene Therapy, 7(8):1206-1206.
10. Akinc A, Thomas M, Klibanov AM, Langer R. 2005. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis[J]. Journal of Gene Medicine, 7(5):657-663.
11. Lu L, Zhu X, Valenzuela RG, Currier BL, Yaszemski MJ. 2001. Biodegradable polymer scaffolds for cartilage tissue engineering[J]. Clinical Orthopaedics and Related Research, (391 Suppl):S251-270.
12. Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. 1998. Polymer concepts in tissue engineering[J]. Journal of Biomedical Materials Research,43(4):422-427.
13. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. 2006. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering[J]. Biomaterials, 27(18):3413-3431.
14. Seunarine K, Gadegaard N, Tormen M, Meredith DO, Riehle MO, Wilkinson CD.2006. 3D polymer scaffolds for tissue engineering[J]. Nanomed, 1(3):281-296.
15. Kallela I, Tulamo RM, Hietanen J, Pohjonen T, Suuronen R, Lindqvist C. 1999. Fixation of mandibular body osteotomies using biodegradable amorphous self-reinforced (70L : 30DL) polylactide or metal lag screws: an experimental study in sheep[J]. Journal of Cranio-Maxillofacial Surgery, 27(2):124-133.
16. Saikku-Backstrom A, Tulamo RM, Pohjonen T, Tormala P, Raiha JE, Rokkanen P. 1999. Material properties of absorbable self-reinforced fibrillated poly-96L/4D-lactide (SR-PLA96) rods; a study in vitro and in vivo[J]. Journal of Materials Science-Materials in Medicine, 10(1):1-8.
17. Savic R, Luo LB, Eisenberg A, Maysinger D. 2003. Micellar nanocontainers distribute to defined cytoplasmic organelles[J]. Science, 300(5619):615-618.
18. Torchilin VP. 2007. Micellar nanocarriers: pharmaceutical perspectives[J]. Pharmaceutical Research, 24(1):1-16.
19. Lee ES, Na K, Bae YH. 2005. Super pH-sensitive multifunctional polymeric micelle[J]. Nano Letters, 5(2):325-329.
20. Lee ES, Gao ZG, Kim D, Park K, Kwon IC, Bae YH. 2008. Super pH-sensitive multifunctional polymeric micelle for tumor pH(e) specific TAT exposure and multidrug resistance[J]. Journal of Controlled Release, 129(3):228-236.
21. Hoffman AS. 2000. Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations[J]. Clinical Chemistry, 46(9):1478-1486.
22. Gil ES, Hudson SA. 2004.Stimuli-reponsive polymers and their bioconjugates[J]. Progress in Polymer Science, 29(12):1173-1222.
23. Rijcken CJF, Soga O, Hennink WE, van Nostrum CF. 2007. Triggered destabilisation of polymeric micelles and vesicles by changing polymers polarity: An attractive tool for drug delivery[J]. Journal of Controlled Release, 120(3):131-148.
24. Liu SQ, Tong YW, Yang YY. 2005. Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) with varying compositions[J]. Biomaterials, 26(24):5064-5074.
25. Lin C, Zhong ZY, Lok MC, et al. 2007. Novel bioreducible poly(amido amine)s for highly efficient gene delivery[J]. Bioconjugate Chemistry, 18(1):138-145.
26. Lin C, Zhong ZY, Lok MC, Jiang XL, Hennink WE, Jan FJ, et al. 2006. Linear poly(amido amine)s with secondary and tertiary amino groups and variable amounts of disulfide linkages: Synthesis and in vitro gene transfer properties[J]. Journal of Controlled Release,28;116(2):130-137.
27. Lin C, Blaauboer CJ, Timoneda MM, et al. 2008 .Bioreducible poly(amido amine)s with oligoamine side chains: synthesis, characterization, and structural effects on gene delivery[J]. Journal of Controlled Release, 3;126(2):166-174.
28. Christensen LV, Chang CW, Kim WJ, et al. 2006. Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery[J]. Bioconjuguate Chemistry, 17(5):1233-1240.
29. Matsumoto S, Christie RJ, Nishiyama N et al. 2009. Environment-Responsive Block Copolymer Micelles with a Disulfide Cross-Linked Core for Enhanced siRNA Delivery[J]. Biomacromolecules,10(1):119-127.
30. Saito G, Swanson JA, Lee KD.2003.Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities[J]. Advanced Drug Delivery Review, 55(2):199-215.
31. Koo AN, Lee HJ, Kim SE, Chang JH, Park C, Kim C, et al. 2008. Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-mediated intracellular drug delivery[J]. Chemical Communications (Camb), Dec 28(48):6570-6572.
32. Xiao CS, Wang YC, Du JZ, Chen XS, Wang J.2006.Kinetics and mechanism of 2-ethoxy-2-oxo-1,3,2-dioxaphospholane polymerization initiated by stannous octoate[J]. Macromolecules, 39(20):6825-6831.
33. Ghosh S, Basu S, Thayumanavan S. 2006. Simultaneous and reversible functionalization of copolymers for biological applications[J]. Macromolecules, 39(17):5595-5597.
34. Carrot G, Hilborn JG, Trollsas M, Hedrick JL. 1999 .Two general methods for the synthesis of thiol-functional polycaprolactones[J]. Macromolecules, 32(16):5264-5269.
35. Du JZ, Chen DP, Wang YC, Xiao CS, Lu YJ, Wang J, et al. 2006. Synthesis and micellization of amphiphilic brush-coil block copolymer based on poly(epsilon-caprolactone) and PEGylated polyphosphoester[J]. Biomacromolecules, 7(6):1898-1903.
36. Li SD, Huang L. 2008. Pharmacokinetics and biodistribution of nanoparticles[J]. Molecular Pharmaceutics, 5(4):496-504.
37. Bae Y, Fukushima S, Harada A, Kataoka K.2003.Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: polymeric micelles that are responsive to intracellular pH change[J]. Angewandte Chemie International Edition, 42(38):4640-4643.
38. Thierry AR, Rabinovich P, Peng B, Mahan LC, Bryant JL, Gallo RC. 1997. Characterization of liposome-mediated gene delivery: Expression, stability and pharmacokinetics of plasmid DNA[J]. Gene Therapy, 4(3):226-237.
39. Sun X, Hai L, Wu Y, Hu HY, Zhang ZR. 2005. Targeted gene delivery to hepatoma cellsusing galactosylated liposome-polycation-DNA complexes (LPD)[J].Journal of Drug Targeting, 13(2):121-128.
40. Park JG, Moon IJ. 2003. Developments of DNA delivery system using the triple complex of DNA: Peptide: Liposome[J]. Molecular Therapy, 7(5):S217-S217.
41. Boulikas T. 1996. Liposome DNA delivery and uptake by cells (review) [J].Oncology Reports, 3(6):989-995.
42. Scaria P, Woodle MC. 2002. Polymer conjugates for gene delivery[M]. Abstracts of Papers of the American Chemical Society, 18;224:U471-U471.
43. Neu M, Germershaus O, Behe M, Kissel T. 2007. Bioreversibly crosslinked polyplexes of PEI and high molecular weight PEG show extended circulation times in vivo[J].Journal of Controlled Release, 124(1-2):69-80.
44. Ahn HH, Lee MS, Cho MH, Shin YN, Lee JH, Kim KS, et al. 2008. DNA/PEI nano-particles for gene delivery of rat bone marrow stem cells[J]. Colloids and Surfaces a-Physicochemical and Engineering Aspects, 313:116-120.
45. Nam YS, Kang HS, Park JY, Park TG, Han SH, Chang IS. 2003. New micelle-like polymer aggregates made from PEI-PLGA diblock copolymers: micellar characteristics and cellular uptake[J]. Biomaterials,24(12):2053-2059.
46. Wang DA, Narang AS, Kotb M, Gaber AO, Miller DD, Kim SW, et al. 2002. Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery[J]. Biomacromolecules, 3(6):1197-1207.
47. Godbey WT, Wu KK, Mikos AG. 1999.Poly(ethylenimine) and its role in gene delivery[J]. Journal of Controlled Release, 60(2-3):149-160.
48. Santos JL, Oramas E, Pego AP, Granja PL, Tomas H. 2009. Osteogenic differentiation of mesenchymal stem cells using PAMAM dendrimers as gene delivery vectors[J]. Journal of Controlled Release, 134(2):141-148.
49. Kim T, Seo HJ, Choi JS, Jang HS, Baek J, Kim K, et al. 2004. PAMAM-PEG-PAMAM: Novel triblock copolymer as a biocompatible and efficient gene delivery carrier[J]. Biomacromolecules, 5(6):2487-2492.
50. Lee JH, Lim YB, Choi JS, Lee Y, Kim TI, Kim HJ, et al. 2003. Polyplexes assembled with internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral surface and gene delivery potency[J]. Bioconjugate Chemistry, 14(6):1214-1221.
51. Erbacher P, Zou SM, Bettinger T, Steffan AM, Remy JS.1998. Chitosan-based vector/DNA complexes for gene delivery: Biophysical characteristics and transfection ability[J]. Pharmaceutical Research, 15(9):1332-1339.
52. Lee KY, Kwon IC, Kim YH, Jo WH, Jeong SY. 1998. Preparation of chitosan self-aggregates as a gene delivery system[J]. Journal of Controlled Release, 51(2-3):213-220.
53. Kim TH, Jiang HL, Nah JW, Cho MH, Akaike T, Cho CS. 2007. Receptor-mediated gene delivery using chitosan derivatives in vitro and in vivo[J].Macromolecular Symposia,249:137-144
54. Strand SP, Issa MM, Christensen BE, Varum KM, Artursson P. 2008.Tailoring of Chitosans for Gene Delivery: Novel Self-Branched Glycosylated Chitosan Oligomers with Improved Functional Properties[J]. Biomacromolecules, 9(11):3268-3276.
55. Mintzer MA, Simanek EE. 2009. Nonviral Vectors for Gene Delivery[J].Chemical Reviews, 109(2):259-302.
56. Forrest ML, Meister GE, Koerber JT, Pack DW. 2004. Partial acetylation of polyethylenimine enhances in vitro gene delivery[J]. Pharmaceutical Research, 21(2):365-371.
57. Rhaese S, von Briesen H, Rubsamen-Waigmann H, Kreuter J, Langer K. 2003. Human serum albumin-polyethylenimine nanoparticles for gene delivery[J].Journal of Controlled Release, 92(1-2):199-208.
58. Carrabino S, Di Gioia S, Copreni E, Conese M. 2005. Serum albumin enhances polyethylenimine-mediated gene delivery to human respiratory epithelial cells[J]. Journal of Gene Medicine, 7(12):1555-1564.
59. Gosselin MA, Guo WJ, Lee RJ. 2001. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine[J]. Bioconjugate Chemistry, 12(6):989-994.
60. Peng Q, Zhong Z, Zhuo R.2008. Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors[J].Bioconjuguate Chemistry,19(2):499-506.
61. Lu B, Xu XD, Zhang XZ, Cheng SX, Zhuo RX. 2008. Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies[J]. Biomacromolecules,9(10):2594-2600.
62. Xiong MP, Forrest ML, Ton G, Zhao A, Davies NM, Kwon GS.2007.Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery[J]. Biomaterials,28(32):4889-4900.
63. Sun YX, Xiao W, Cheng SX, Zhang XZ, Zhuo RX. 2008. Synthesis of (Dex-HMDI)-g-PEIs as effective and low cytotoxic nonviral gene vectors[J]. Journal of Controlled Release, 4;128(2):171-178.
64. Thomas M, Klibanov AM. 2002. Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 99(23):14640-14645.
65. Doody AM, Korley JN, Dang KP, Zawaneh PN, Putnam D. 2006. Characterizing the structure/function parameter space of hydrocarbon-conjugated branched polyethylenimine for DNA delivery in vitro[J]. Journal of Controlled Release, 116(2):227-237.
66. Zintchenko A, Philipp A, Dehshahri A, Wagner E. 2008. Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity[J].Bioconjugate Chemistry, 19(7):1448-1455.
67. Sun TM, Du JZ, Yan LF, Mao HQ, Wang J. 2008. Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery[J]. Biomaterials, 29(32):4348-4355.
68. Wang YC, Tang LY, Li Y, Wang J.2009.Thermoresponsive Block Copolymers of Poly(ethylene glycol) and Polyphosphoester: Thermo-Induced Self-Assembly, Biocompatibility, and Hydrolytic Degradation[J]. Biomacromolecules, 10(1):66-73.
69. Ashkenazi N, Zade SS, Segall Y, Karton Y, Bendikov M. 2005. Selective site controlled nucleophilic attacks in 5-membered ring phosphate esters: unusual C-O vs. common P-O bond cleavage[J]. Chemical Communications, (47):5879-5881.
70. Jiang X, Dai H, Ke CY, Mo X, Torbenson MS, Li Z, et al. 2007. PEG-b-PPA/DNA micelles improve transgene expression in rat liver through intrabiliary infusion[J]. Journal of Control Release, 122(3):297-304.
71. Xu DM, Yao SD, Liu YB, Sheng KL, Hong J, Gong PJ, et al. 2007. Size-dependent properties of M-PEIs nanogels for gene delivery in cancer cells[J]. International Journal of Pharmaceutics, 338(1-2):291-296.
72. Tian HY, Xiong W, Wei JZ, Wang Y, Chen XS, Jing XB, et al. 2007. Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG[J]. Biomaterials,28(18):2899-2907.
73. Kunath K, von Harpe A, Fischer D, Peterson H, Bickel U, Voigt K, et al. 2003. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine[J]. Journal of Controlled Releas, 14;89(1):113-125.
74. Jiang X, Lok MC, Hennink WE. 2007. Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery[J]. Bioconjugate Chemistry, 18(6):2077-2084.