功能性嵌段聚合物的设计与合成
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摘要
追求药物的高活性、低毒性、良好的物理化学性质、优越的药代动力学性质以及病人良好的顺应性,是众多药物研发工作者努力的目标。然而,一个传统的化学实体很难实现同时具有高效、低毒、缓释、透膜和靶向性等多种功能。随着生命科学和高分子科学的发展,以高分子材料为载体的药物释放系统以及高分子偶联药物开始成为人们关注的热点。两种学科的紧密联系和交叉发展开启了“polymer therapeutics”的新时代,众多的低毒或无毒的高分子材料被应用到药物研发领域,高分子材料已逐渐由辅助作用向主导地位转变,形成了具有特征的高分子药物和新型药物制剂,为药物的发展提供了更为广阔的空间,也为多功能药物实体的研发奠定了重要的基础,使获得多功能药物实体成为可能。
本课题设想制备一个球形的药物实体——多功能磁性纳米微球药物实体,将一些功能配基(药物分子、生物靶向分子、透膜物质等)通过共价键分别连接到聚合物上,再将这些具有相同络合能力的功能聚合物按照设计比例与磁性颗粒络合以制备稳定的磁性纳米微球,获得具有多功能协同作用的磁性纳米微球药物实体。本文是“多功能磁性纳米微球药物实体”课题的一部分,主要是设计并制备连有活性药物、靶向物质、透膜肽等功能分子的嵌段聚合物,为多功能药物实体模型建立提供性质优良的、具有功能基团的聚合物。如何将功能基团与嵌段聚合物键连是本课题的难点;制备连有药物、靶向物质、透膜肽片段等基团的嵌段聚合物并进行表征,是本文的重点。通过文献调研和课题组前期工作,本文选用具有良好生物相容性且代谢安全的聚乙二醇-b-聚丙烯酰甘油单酯(PEG-b-PGA)嵌段聚合物为稳定磁性微球的载体。
本文以PEG600为实验的起始原料,对其进行端基修饰,获得一端连有叠氮基,另一端连有溴原子的异官能团PEG修饰剂;以结构简单、易于修饰的抗肿瘤药物5-氟尿嘧啶为模型药物;以具有肝靶向性的半乳糖作为靶向物质;以具有透膜功能的Tat(48-57)短肽序列作为透膜物质。根据各功能分子的结构特点,分别设计路线使其键连到嵌段聚合物中。
在制备连有药物分子的嵌段聚合物过程中,本文先后设计了多条合成路线,最终确定了两条最优路线,并且两条路线中都应用到了Click反应和原子转移自由基聚合反应(ATRP)。Click反应是近年来研究者新发现的一种反应类型,在一价铜存在的条件下,叠氮基与炔基可以发生1,3-偶极加成反应,生成稳定的五元氮唑环,该反应具有反应条件温和,高选择性和定量的特点。Click反应的引入为复杂结构高分子的设计提供了便捷高效且实用性强的方法,为高分子的合成和构造开辟了一条新途径。
第一条路线是将氟尿嘧啶分子炔基衍生化后与PEG分子一端的叠氮基进行Click反应,制备大分子引发剂,再通过ATRP反应,可得到分子量可控且分布较窄的连有氟尿嘧啶药物分子的聚乙二醇-b-聚丙烯酰甘油缩醛(PEG-b-PSA)。在合成的过程中对每一个产物都进行了1H NMR检测,并用凝胶渗透色谱(GPC)对聚合物的分子量进行测试。在对聚合物缩醛基团脱保护后获得连有氟尿嘧啶药物分子的PEG-b-PGA嵌段聚合物。这条路线为制备其他功能大分子奠定了基础,但是,由于不同大分子引发剂的活性差异较大,使得各批次间聚合的分子量也不同。利用此种方法要获得具有相同分子量的三类功能聚合物存在很大困难。为此需要进行其他路线的探索。
本文利用马来酰亚胺基中的双键可以与巯基进行定量反应的特性,设计合成了第二条路线:以马来酰亚胺基为端基的嵌段共聚物以及氟尿嘧啶的巯基衍生物。先用呋喃环对马来酰胺端基的双键进行保护,以防止双键参与聚合形成交联,再进行炔基衍生化。然后与PEG修饰剂进行Click反应制备大分子引发剂,通过ATRP聚合获得呋喃保护的马来酰亚胺基PEG-b-PSA,在合成的过程中对每一个产物都进行了1H NMR检测,并对聚合物的分子量进行了GPC测试。在一定条件下脱除呋喃环和缩醛保护基后,聚合物可以在水中与巯基化的氟尿嘧啶发生反应制备出连有氟尿嘧啶分子的嵌段聚合物。
在第二条路线的启发下,本文设计并制备出了半乳糖的巯基衍生物,用相同的方法将半乳糖以及连有半胱氨酸的透膜肽片段定点连接到嵌段聚合物的马来酰亚胺端基上。最终获得三种具有相同分子量的并分别连有氟尿嘧啶基、半乳糖配基、透膜肽片段的PEG-b-PGA,为多功能磁性纳米微球的制备以及课题深入开展打下了坚实的基础。
本课题组已将氟尿嘧啶基-PEG-b-PGA与Fe3O4络合制备了载药磁性纳米微球,透射电镜检测粒径在20nm左右,同时对微球的稳定性进行了评价,其水溶液可以稳定分散半年以上,得到了较为理想的结果。载有其它功能基团的磁性纳米微球正在制备之中,相关动物实验也在开展。
Improving the therapeutic index of drugs is a major impetus for innovation in many therapeutic areas. The ideal drug is hoped to have some good properties, such as: high activity, low toxicity, good chemical and physical properties, and so on. But it is very difficult for a traditional drug to possess so many good characters by itself.
With the development and cooperation of pharmacy, chemistry and polymer science, a new era of polymer therapeutics comes. Lots of good polymers have been used in drug research. The development of synthetic polymer drugs and therapeutics make it possible to construct a new chemical entity that can possesses all desired properties of drugs. In order to solve those problem, our group propose to synthesis a multifunctional drug entity which have desirable properties. The entity is composed of magnetic nanosphere and different functional copolymers. In this entity, good efficacy drugs and several different ligands with special functions are conjugated to one end of the block copolymers respectively, while the other end of the polymer are used to complex with Fe3O4 particles.
In this paper, we are focusing on the synthesis of functional copolymers with different drugs and functional ligands. Basing on the former work, we choose PEG-b-PGA as the block copolymer, 5-fluorouracil as the modifying drug, galactose as the targeting ligand, and Tat (48-57) as cell penetrating peptide. At the beginning of research, we synthesized heterobifunctional PEG600 as one of the raw materials of the following research, which has bromide on one end and azide group on the other end.
During the research, we designed two routes to synthesize drug conjugated block copolymers. One is to synthesize macromolecular initiator containing fluorouracil by click reaction first, and then using the initiator to synthesize 5-Fluorouracil- PEG-b-PGA by ATRP reaction. The other one is to synthesize furan protected Maleimido-PEG-b-PGA and thiol derivatives of 5-fluorouracil and galactose respectively. Then we can get Maleimido-PEG-b-PGA by deprotecting the furan ring. In the following step, we use different thiol derivatives to react with block copolymer, and can get 5-Fluorouracil-Maleimido-PEG-b-PGA, Galactose-Maleimido-PEG-b- PGA and Tat-Maleimido-PEG-b-PGA. We prefer to use the second route to synthesize functional block copolymer, because we can get different functional polymers by using the same copolymer and it can lessen the effect of molecular weight. During the experiment, the reaction process is monitored by HPLC and the structures of products are all identified by 1H NMR. The molecular weight of the final functional block copolymers is assessed by GPC.
The results of GPC show that the molecular weights of functional PEG-b-PGA synthesized by ATRP reaction are controllable and have a narrow distribution. The magnetic nanosphere complexation with 5-Fluorouracil-PEG-b-PGA has been prepared and the stability of nanosphere has been investigated. Its water solution can stabilize more than half a year. The animal experiment will be developed soon.
本课题设想制备一个球形的药物实体——多功能磁性纳米微球药物实体,将一些功能配基(药物分子、生物靶向分子、透膜物质等)通过共价键分别连接到聚合物上,再将这些具有相同络合能力的功能聚合物按照设计比例与磁性颗粒络合以制备稳定的磁性纳米微球,获得具有多功能协同作用的磁性纳米微球药物实体。本文是“多功能磁性纳米微球药物实体”课题的一部分,主要是设计并制备连有活性药物、靶向物质、透膜肽等功能分子的嵌段聚合物,为多功能药物实体模型建立提供性质优良的、具有功能基团的聚合物。如何将功能基团与嵌段聚合物键连是本课题的难点;制备连有药物、靶向物质、透膜肽片段等基团的嵌段聚合物并进行表征,是本文的重点。通过文献调研和课题组前期工作,本文选用具有良好生物相容性且代谢安全的聚乙二醇-b-聚丙烯酰甘油单酯(PEG-b-PGA)嵌段聚合物为稳定磁性微球的载体。
本文以PEG600为实验的起始原料,对其进行端基修饰,获得一端连有叠氮基,另一端连有溴原子的异官能团PEG修饰剂;以结构简单、易于修饰的抗肿瘤药物5-氟尿嘧啶为模型药物;以具有肝靶向性的半乳糖作为靶向物质;以具有透膜功能的Tat(48-57)短肽序列作为透膜物质。根据各功能分子的结构特点,分别设计路线使其键连到嵌段聚合物中。
在制备连有药物分子的嵌段聚合物过程中,本文先后设计了多条合成路线,最终确定了两条最优路线,并且两条路线中都应用到了Click反应和原子转移自由基聚合反应(ATRP)。Click反应是近年来研究者新发现的一种反应类型,在一价铜存在的条件下,叠氮基与炔基可以发生1,3-偶极加成反应,生成稳定的五元氮唑环,该反应具有反应条件温和,高选择性和定量的特点。Click反应的引入为复杂结构高分子的设计提供了便捷高效且实用性强的方法,为高分子的合成和构造开辟了一条新途径。
第一条路线是将氟尿嘧啶分子炔基衍生化后与PEG分子一端的叠氮基进行Click反应,制备大分子引发剂,再通过ATRP反应,可得到分子量可控且分布较窄的连有氟尿嘧啶药物分子的聚乙二醇-b-聚丙烯酰甘油缩醛(PEG-b-PSA)。在合成的过程中对每一个产物都进行了1H NMR检测,并用凝胶渗透色谱(GPC)对聚合物的分子量进行测试。在对聚合物缩醛基团脱保护后获得连有氟尿嘧啶药物分子的PEG-b-PGA嵌段聚合物。这条路线为制备其他功能大分子奠定了基础,但是,由于不同大分子引发剂的活性差异较大,使得各批次间聚合的分子量也不同。利用此种方法要获得具有相同分子量的三类功能聚合物存在很大困难。为此需要进行其他路线的探索。
本文利用马来酰亚胺基中的双键可以与巯基进行定量反应的特性,设计合成了第二条路线:以马来酰亚胺基为端基的嵌段共聚物以及氟尿嘧啶的巯基衍生物。先用呋喃环对马来酰胺端基的双键进行保护,以防止双键参与聚合形成交联,再进行炔基衍生化。然后与PEG修饰剂进行Click反应制备大分子引发剂,通过ATRP聚合获得呋喃保护的马来酰亚胺基PEG-b-PSA,在合成的过程中对每一个产物都进行了1H NMR检测,并对聚合物的分子量进行了GPC测试。在一定条件下脱除呋喃环和缩醛保护基后,聚合物可以在水中与巯基化的氟尿嘧啶发生反应制备出连有氟尿嘧啶分子的嵌段聚合物。
在第二条路线的启发下,本文设计并制备出了半乳糖的巯基衍生物,用相同的方法将半乳糖以及连有半胱氨酸的透膜肽片段定点连接到嵌段聚合物的马来酰亚胺端基上。最终获得三种具有相同分子量的并分别连有氟尿嘧啶基、半乳糖配基、透膜肽片段的PEG-b-PGA,为多功能磁性纳米微球的制备以及课题深入开展打下了坚实的基础。
本课题组已将氟尿嘧啶基-PEG-b-PGA与Fe3O4络合制备了载药磁性纳米微球,透射电镜检测粒径在20nm左右,同时对微球的稳定性进行了评价,其水溶液可以稳定分散半年以上,得到了较为理想的结果。载有其它功能基团的磁性纳米微球正在制备之中,相关动物实验也在开展。
Improving the therapeutic index of drugs is a major impetus for innovation in many therapeutic areas. The ideal drug is hoped to have some good properties, such as: high activity, low toxicity, good chemical and physical properties, and so on. But it is very difficult for a traditional drug to possess so many good characters by itself.
With the development and cooperation of pharmacy, chemistry and polymer science, a new era of polymer therapeutics comes. Lots of good polymers have been used in drug research. The development of synthetic polymer drugs and therapeutics make it possible to construct a new chemical entity that can possesses all desired properties of drugs. In order to solve those problem, our group propose to synthesis a multifunctional drug entity which have desirable properties. The entity is composed of magnetic nanosphere and different functional copolymers. In this entity, good efficacy drugs and several different ligands with special functions are conjugated to one end of the block copolymers respectively, while the other end of the polymer are used to complex with Fe3O4 particles.
In this paper, we are focusing on the synthesis of functional copolymers with different drugs and functional ligands. Basing on the former work, we choose PEG-b-PGA as the block copolymer, 5-fluorouracil as the modifying drug, galactose as the targeting ligand, and Tat (48-57) as cell penetrating peptide. At the beginning of research, we synthesized heterobifunctional PEG600 as one of the raw materials of the following research, which has bromide on one end and azide group on the other end.
During the research, we designed two routes to synthesize drug conjugated block copolymers. One is to synthesize macromolecular initiator containing fluorouracil by click reaction first, and then using the initiator to synthesize 5-Fluorouracil- PEG-b-PGA by ATRP reaction. The other one is to synthesize furan protected Maleimido-PEG-b-PGA and thiol derivatives of 5-fluorouracil and galactose respectively. Then we can get Maleimido-PEG-b-PGA by deprotecting the furan ring. In the following step, we use different thiol derivatives to react with block copolymer, and can get 5-Fluorouracil-Maleimido-PEG-b-PGA, Galactose-Maleimido-PEG-b- PGA and Tat-Maleimido-PEG-b-PGA. We prefer to use the second route to synthesize functional block copolymer, because we can get different functional polymers by using the same copolymer and it can lessen the effect of molecular weight. During the experiment, the reaction process is monitored by HPLC and the structures of products are all identified by 1H NMR. The molecular weight of the final functional block copolymers is assessed by GPC.
The results of GPC show that the molecular weights of functional PEG-b-PGA synthesized by ATRP reaction are controllable and have a narrow distribution. The magnetic nanosphere complexation with 5-Fluorouracil-PEG-b-PGA has been prepared and the stability of nanosphere has been investigated. Its water solution can stabilize more than half a year. The animal experiment will be developed soon.
引文
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[1] Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discovery Today, 2005, 10(21): 1451-1458.
[2] Schwabacher AW, Lane JW, Schiesher MW,Leigh KM, Johnson CW. Desymmetrization Reactions: Efficient Preparation of Unsymmetrically Substituted Linker M olecules. J. Org. Chem., 1998, 63: 1727-1729.
[3] Thompson MS, Vadala TP, Vadala ML, Lin Y, Riffle JS. synthesis and applications of heterobifunctional poly(ethylene oxide) oligomers. Polymer, 2008, 49: 345-373.
[4] Li J, Kao WJ. Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated- Peptide Biopolymer Conjugates, Biomacromolecules, 2003, 4: 1055-1067.
[5] Lyer SS, Anderson AS, Reed S, Swanson B, and Schmidt JG. Synthesis of orthogonal end functionalized oligoethylene glycols of defined lengths. Tetrahedron Letters, 2004, 45: 4285-4288.
[6] Working PK, Newman MS, Johnson J. Safety of poly(ethylene glycol) and poly(ethylene glycol) derivatives, in: J.M. Harris, S. Zalipsky (Eds.). Poly(ethylene glycol) Chemistry and Biological Application, ACS Books, Washington, DC, 1997, pp45-57.
[7] Fazio F, Bryan MC, Blixt O. Synthesis of Sugar Arrays in Microtiter Plate. J. Am. Chem. Soc., 2002, 124: 14397-14402.
[8] Ladmiral V, Mantovani G, Clarkson GJ. Synthesis of Neoglycopolymers by a Combination of “Click Chemistry” and Living Radical Polymerization. J. Am. Chem. Soc., 2006, 128: 4823-4830.
[9] Hetet CL, David M, Carreaux F, Carboni B, Sauleau A. Synthesis of functionalized γ- and δ-lactones via polymer-bound epoxides. Tetrahedron Letters, 1997, 38(29): 5153-5156.
[10] Tsarevsky NV, Sumerlin BS, Matyjaszewski K. Step-Growth “Click” Coupling of Telechelic Polymers Prepared by Atom Transfer Radical Polymerization. Macromolecules, 2005, 38: 3558-3561.
[11] Wu P, Feldman AK, Nugent AK, Hawker CJ, Scheel A, Voit B, Pyun J, Fréchet JM, Sharpless KB, Fokin VV. Efficiency and Fidelity in a Click-Chemistry Route to Triazole Dendrimers by the Copper(I)-Catalyzed Ligation of Azides and Alkynes. Angew. Chem. Int. Ed., 2004, 43: 3928-3932.
[12] Sun XL, Stabler CL, Cazalis CS, and Chaikof EL. Carbohydrate and Protein Immobilization onto Solid Surfaces by Sequential Diels-Alder and Azide-Alkyne Cycloadditions. Bioconjugate Chem., 2006, 17: 52-57.
[13] Xu CY, Huang MZ. An Approach to New Water-soluble Oligo(ethylene glycol) Camptothecin Analogues by 1,3-Dipolar Cycloaddition. Chinese Chemical Letters, 2006, 17(7): 883-886.
[14] Lutz JF, B?rner HG, Weichenhan K. Combining Atom Transfer Radical Polymerization and Click Chemistry: A Versatile Method for the Preparation of End-Functional Polymers. Macromol. Rapid Commun., 2005, 26: 514-518.
[15] Bock VD, Hiemstra H, Maarseveen JH. CuI-Catalyzed Alkyne-Azide“Click” Cycloadditions from a Mechanistic and Synthetic Perspective. Eur. J. Org. Chem., 2006, 1:51-68.
[16] Matyjaszewsski K, Xia JH. Atom Transfer Radical Polymerization. Chem. Rev., 2001, 101: 2921-2990.
[17] 周其凤,胡汉杰,高分子化学,化学工业出版社,2001 年版。
[18] Mantovani G, Ladmiral V, Tao L, Haddleton DM. One-pot tandem living radical polymerization-Huisgens cycloaddition process (“Click”) catalyzed by N-alkyl-2- pyridylmethanimine/Cu(I)Br complexes. Chem. Commun., 2005, 2089-2091.
[19] Eggelte TA, Koning H, Huisman HO. Synthesis and some reductions of endo- and exo-3,6-epoxy- Δ 4-tetrahydrophthalic anhydride. Tetrahedron, 1973, 29(16): 2445-2447.
[20] Bolm C, Dinter C, Seger A, H?cker H, Brozio J. Synthesis of Catalytically Active Polymers by means of ROMP: An Effective Approach towards Polymeric Homogeneously Soluble Catalysts. J. Org. Chem., 1999, 64(16): 5730-5731.
[21] Cho YW, Lee JR, Song SC. Novel Thermosensitive 5-Fluorouracil-Cyclotriphosphazene Conjugates: Synthesis, Thermosensitivity, Degradability, and in Vitro Antitumor Activity. Bioconjug. Chem., 2005, 16: 1529-1535.
[22] Seely J, Richey C, Grasel T. Issues encountered in the production of site specific mono-PEGylated therapeutic protein. Polym. Prepr(Am. Chem. Soc., Div. Polym. Chem.), 1997, 38(1): 572-573.
[23] Garnett MC. Targeted drug conjugates: principles and progress. Adv.Drug Rev., 2001, 53: 171-216.
[24] Wall DA, Wilson G, Hubbard AL. The galactose specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell, 1980, 21: 79293.
[25] Junrichi M, Yuichi O, Tatsuro O. Possibility of application of quaternary chitosan having pendant galactose residues as gene delivery tool. Carbohydr. Polym., 1996, 29: 69274.
[26] Gao SY, Chen JN, Xu XR , Ding Z, Yang YH, Hua ZC, Zhang JF. Galactosylated low molecular weight chitosan as DNA carrier for hepatocyte-targeting. In. J. Pharm., 2003, 255: 57268.
[27] Park YK, Park YH, Shin BA, Choi ES, Park YR, Akaike T,Cho CS. Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier. J. Control. Rel., 2000, 69: 97-108.
[28] Park IK, Kim TH, Park YH, Shin BA, Choi ES, Chowdhury EH, Akaike T,Cho CS. Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA carrier. J. Control. Rel., 2001, 76: 349-362.
[29] Park IK, Ihm JE, Park YH, Choi YJ, Kim SI, Kim WJ, Akaike T, Cho CS. Galactosylated chitosan (GC)-graft-poly (vinyl pyrrolidone) (PVP) as hepatocyte- targeting DNA carrier. J. Control. Rel., 2003, 86: 349-361.
[30] Beverley T, Carolina HA. Synthetic polymers as drugs and therapeutics. J. Mater. Chem., 2005, 15: 441-455.
[31] Wilkinson BL, Bornaghi LF, Poulsen SA, Houston TA. Synthetic utility of glycosyl triazoles in carbohydrate chemistry. Tetrahedron, 2006, 62: 8115-8125.
[32] Matja? Zorko, ?lo Langel. Cell-penetrating peptides: mechanism and kineticsof cargo delivery. Advanced Drug Deliv. Rev., 2005, 57: 529-545.
[33] Aparna Nori, Jind?ich Kopecek. Intracellular targeting of polymer-bound drugs for cancer chemotherapy. Adv. Drug Deliv. Rev., 2005, 57: 609– 636.
[34] Schwarze SR, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science, 1999, 3: 1569-1572.
[35] Nori A, Jensen KD, Tijerina M. Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery to Human Ovarian Carcinoma Cells. Bioconjug. Chem., 2003, 14: 4450.
[36] Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol., 2000, 18: 410-414.
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[29] Zorko M, Langel ?. Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv. Drug Deliv. Rev., 2005, 57: 529– 545.
[30] Nori A, Kope?ek J. Intracellular targeting of polymer-bound drugs for cancer chemotherapy. Adv. Drug Deliv. Rev., 2005, 57: 609– 636.
[31] Nori A, Jensen KD, Tijerina M. Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery To Human Ovarian Carcinoma Cells. Bioconjug. Chem., 2003, 14: 4450.
[1] Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discovery Today, 2005, 10(21): 1451-1458.
[2] Schwabacher AW, Lane JW, Schiesher MW,Leigh KM, Johnson CW. Desymmetrization Reactions: Efficient Preparation of Unsymmetrically Substituted Linker M olecules. J. Org. Chem., 1998, 63: 1727-1729.
[3] Thompson MS, Vadala TP, Vadala ML, Lin Y, Riffle JS. synthesis and applications of heterobifunctional poly(ethylene oxide) oligomers. Polymer, 2008, 49: 345-373.
[4] Li J, Kao WJ. Synthesis of Polyethylene Glycol (PEG) Derivatives and PEGylated- Peptide Biopolymer Conjugates, Biomacromolecules, 2003, 4: 1055-1067.
[5] Lyer SS, Anderson AS, Reed S, Swanson B, and Schmidt JG. Synthesis of orthogonal end functionalized oligoethylene glycols of defined lengths. Tetrahedron Letters, 2004, 45: 4285-4288.
[6] Working PK, Newman MS, Johnson J. Safety of poly(ethylene glycol) and poly(ethylene glycol) derivatives, in: J.M. Harris, S. Zalipsky (Eds.). Poly(ethylene glycol) Chemistry and Biological Application, ACS Books, Washington, DC, 1997, pp45-57.
[7] Fazio F, Bryan MC, Blixt O. Synthesis of Sugar Arrays in Microtiter Plate. J. Am. Chem. Soc., 2002, 124: 14397-14402.
[8] Ladmiral V, Mantovani G, Clarkson GJ. Synthesis of Neoglycopolymers by a Combination of “Click Chemistry” and Living Radical Polymerization. J. Am. Chem. Soc., 2006, 128: 4823-4830.
[9] Hetet CL, David M, Carreaux F, Carboni B, Sauleau A. Synthesis of functionalized γ- and δ-lactones via polymer-bound epoxides. Tetrahedron Letters, 1997, 38(29): 5153-5156.
[10] Tsarevsky NV, Sumerlin BS, Matyjaszewski K. Step-Growth “Click” Coupling of Telechelic Polymers Prepared by Atom Transfer Radical Polymerization. Macromolecules, 2005, 38: 3558-3561.
[11] Wu P, Feldman AK, Nugent AK, Hawker CJ, Scheel A, Voit B, Pyun J, Fréchet JM, Sharpless KB, Fokin VV. Efficiency and Fidelity in a Click-Chemistry Route to Triazole Dendrimers by the Copper(I)-Catalyzed Ligation of Azides and Alkynes. Angew. Chem. Int. Ed., 2004, 43: 3928-3932.
[12] Sun XL, Stabler CL, Cazalis CS, and Chaikof EL. Carbohydrate and Protein Immobilization onto Solid Surfaces by Sequential Diels-Alder and Azide-Alkyne Cycloadditions. Bioconjugate Chem., 2006, 17: 52-57.
[13] Xu CY, Huang MZ. An Approach to New Water-soluble Oligo(ethylene glycol) Camptothecin Analogues by 1,3-Dipolar Cycloaddition. Chinese Chemical Letters, 2006, 17(7): 883-886.
[14] Lutz JF, B?rner HG, Weichenhan K. Combining Atom Transfer Radical Polymerization and Click Chemistry: A Versatile Method for the Preparation of End-Functional Polymers. Macromol. Rapid Commun., 2005, 26: 514-518.
[15] Bock VD, Hiemstra H, Maarseveen JH. CuI-Catalyzed Alkyne-Azide“Click” Cycloadditions from a Mechanistic and Synthetic Perspective. Eur. J. Org. Chem., 2006, 1:51-68.
[16] Matyjaszewsski K, Xia JH. Atom Transfer Radical Polymerization. Chem. Rev., 2001, 101: 2921-2990.
[17] 周其凤,胡汉杰,高分子化学,化学工业出版社,2001 年版。
[18] Mantovani G, Ladmiral V, Tao L, Haddleton DM. One-pot tandem living radical polymerization-Huisgens cycloaddition process (“Click”) catalyzed by N-alkyl-2- pyridylmethanimine/Cu(I)Br complexes. Chem. Commun., 2005, 2089-2091.
[19] Eggelte TA, Koning H, Huisman HO. Synthesis and some reductions of endo- and exo-3,6-epoxy- Δ 4-tetrahydrophthalic anhydride. Tetrahedron, 1973, 29(16): 2445-2447.
[20] Bolm C, Dinter C, Seger A, H?cker H, Brozio J. Synthesis of Catalytically Active Polymers by means of ROMP: An Effective Approach towards Polymeric Homogeneously Soluble Catalysts. J. Org. Chem., 1999, 64(16): 5730-5731.
[21] Cho YW, Lee JR, Song SC. Novel Thermosensitive 5-Fluorouracil-Cyclotriphosphazene Conjugates: Synthesis, Thermosensitivity, Degradability, and in Vitro Antitumor Activity. Bioconjug. Chem., 2005, 16: 1529-1535.
[22] Seely J, Richey C, Grasel T. Issues encountered in the production of site specific mono-PEGylated therapeutic protein. Polym. Prepr(Am. Chem. Soc., Div. Polym. Chem.), 1997, 38(1): 572-573.
[23] Garnett MC. Targeted drug conjugates: principles and progress. Adv.Drug Rev., 2001, 53: 171-216.
[24] Wall DA, Wilson G, Hubbard AL. The galactose specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell, 1980, 21: 79293.
[25] Junrichi M, Yuichi O, Tatsuro O. Possibility of application of quaternary chitosan having pendant galactose residues as gene delivery tool. Carbohydr. Polym., 1996, 29: 69274.
[26] Gao SY, Chen JN, Xu XR , Ding Z, Yang YH, Hua ZC, Zhang JF. Galactosylated low molecular weight chitosan as DNA carrier for hepatocyte-targeting. In. J. Pharm., 2003, 255: 57268.
[27] Park YK, Park YH, Shin BA, Choi ES, Park YR, Akaike T,Cho CS. Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier. J. Control. Rel., 2000, 69: 97-108.
[28] Park IK, Kim TH, Park YH, Shin BA, Choi ES, Chowdhury EH, Akaike T,Cho CS. Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA carrier. J. Control. Rel., 2001, 76: 349-362.
[29] Park IK, Ihm JE, Park YH, Choi YJ, Kim SI, Kim WJ, Akaike T, Cho CS. Galactosylated chitosan (GC)-graft-poly (vinyl pyrrolidone) (PVP) as hepatocyte- targeting DNA carrier. J. Control. Rel., 2003, 86: 349-361.
[30] Beverley T, Carolina HA. Synthetic polymers as drugs and therapeutics. J. Mater. Chem., 2005, 15: 441-455.
[31] Wilkinson BL, Bornaghi LF, Poulsen SA, Houston TA. Synthetic utility of glycosyl triazoles in carbohydrate chemistry. Tetrahedron, 2006, 62: 8115-8125.
[32] Matja? Zorko, ?lo Langel. Cell-penetrating peptides: mechanism and kineticsof cargo delivery. Advanced Drug Deliv. Rev., 2005, 57: 529-545.
[33] Aparna Nori, Jind?ich Kopecek. Intracellular targeting of polymer-bound drugs for cancer chemotherapy. Adv. Drug Deliv. Rev., 2005, 57: 609– 636.
[34] Schwarze SR, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science, 1999, 3: 1569-1572.
[35] Nori A, Jensen KD, Tijerina M. Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery to Human Ovarian Carcinoma Cells. Bioconjug. Chem., 2003, 14: 4450.
[36] Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol., 2000, 18: 410-414.