MPEG-树枝状聚天冬氨酸两亲性嵌段共聚物的制备及性能研究
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摘要
本文首先以天然天冬氨酸为原料,分别对其氨基和羧基进行保护,得到叔丁氧羰基保护氨基和酯基保护羧基的天冬氨酸,然后采用液相合成法合成了树枝状二代和三代聚天冬氨酸乙酯,再用端羧基聚乙二醇单甲醚(Mn=5000)与树枝状三代聚天冬氨酸乙酯脱水缩合得到MPEG-树枝状三代聚天冬氨酸两亲性嵌段共聚物,端酯基水解,酸化后分别与树枝状二代和三代聚天冬氨酸乙酯脱水缩合,最终得到了MPEG-树枝状五代和六代聚天冬氨酸两亲嵌段共聚物,并利用凝胶渗透色谱(GPC)、红外光谱(FT-IR)、示差扫描量热法(DSC)表征了最终产物。将MPEG-树枝状五代六代聚天冬氨酸两亲性嵌段共聚物在水中进行自组装,测定了两者的临界胶束浓度,并用核磁共振氢谱(’H-NMR),动态光散射(DLS)和扫描电镜(SEM)研究其自组装形成的胶束结构以及胶束的粒径。以甲氨蝶呤为模型药物,以两亲性嵌段共聚物聚乙二醇-树枝状聚天冬氨酸为载体,制备聚乙二醇-聚天冬氨酸载药纳米胶束,探讨其结构、理化性质和体外释药研究,旨在提高药物的疗效和降低毒性,开拓纳米载药胶束体系应用的新方向,为发展新一代的纳米载药胶束体系提供科学依据。
First of all, Boc-Asp-COOH and ASP-COOC2H5 was synthesized by using natural L-aspartic acid. Then, G2.0 and G3.0 dendritic Poly(aspartic acid)s ethylester were obtained through liquid-phase synthesis method. MPEG-COOH (Mn=5000) and G3.0 dendritic Poly(aspartic acid)s ethylester dehydrated to get G3.0 MPEG-dendritic Poly(aspartic acid)s ethylester which would be hydrolysised and acidified. G3.0 MPEG-dendritic Poly(aspartic acid)s reacted with G2.0 and G3.0 dendritic Poly(aspartic acid)s ethylester separately and given to the target polymers G5.0 and G6.0 MPEG-dendritic Poly(aspartic acid)s ethylester. The polymers were characterized by1H-NMR, GPC, FT-IR and DSC. Compared with the solubility of polymers with different generation, CMC were measured and the micelles self-assembled by G5.0 and G6.0 MPEG-dendritic Poly(aspartic acid)s ethylester were studied on stability and distribution of particle size by means of1-NMR, DLS and SEM detection. MTX was uploaded by MPEG-dendritic Poly(aspartic acid)s ethylester micelles and the structure, physicochemical properties and drug release behavior were studied, in order to improve drug efficacy and reduced toxicity, for the purpose of developing new polymer micelle drug carrier systems.
First of all, Boc-Asp-COOH and ASP-COOC2H5 was synthesized by using natural L-aspartic acid. Then, G2.0 and G3.0 dendritic Poly(aspartic acid)s ethylester were obtained through liquid-phase synthesis method. MPEG-COOH (Mn=5000) and G3.0 dendritic Poly(aspartic acid)s ethylester dehydrated to get G3.0 MPEG-dendritic Poly(aspartic acid)s ethylester which would be hydrolysised and acidified. G3.0 MPEG-dendritic Poly(aspartic acid)s reacted with G2.0 and G3.0 dendritic Poly(aspartic acid)s ethylester separately and given to the target polymers G5.0 and G6.0 MPEG-dendritic Poly(aspartic acid)s ethylester. The polymers were characterized by1H-NMR, GPC, FT-IR and DSC. Compared with the solubility of polymers with different generation, CMC were measured and the micelles self-assembled by G5.0 and G6.0 MPEG-dendritic Poly(aspartic acid)s ethylester were studied on stability and distribution of particle size by means of1-NMR, DLS and SEM detection. MTX was uploaded by MPEG-dendritic Poly(aspartic acid)s ethylester micelles and the structure, physicochemical properties and drug release behavior were studied, in order to improve drug efficacy and reduced toxicity, for the purpose of developing new polymer micelle drug carrier systems.
引文
[1]龙华丽,张钧寿,张瑛.聚乙二醇脂肪酸甘油酯在药剂学上的应用[J].药学进展,2004,28(6)：262-269.
[2]张大为.功能化高分子胶束药物载体的制备与研究[学].北京化工大学,2010.
[3]Amedo A, lrache J M, Merodio M, et al. AlbuIllin nanopmiclesimproved the stability, nuclear accumulation and anticytomegaloviral activity of aphosphodiester oligonucleotide[J]. Controlled Release,2004,94(1):217.
[4]可伟,杨卓理,孙玉峰,刘艳.大分子胶束作为药物载体的应用[J].中国新药杂志,2006,15(19)：1630-1635.
[5]Allen C, Maysinger D, EisenbcIg A. Nano. engineering block copolymer aggregates for drug delivery[J]. colloids SurfB,1999,16:1-35.
[6]Yu B G, Okano T, Kataoka K, et al. Polymeric micelle for drug delivery; solubilizafion and haemoiytie activity of amphotericin B[J]. J Controlled Release, 1998,53(1-3):131-136.
[7]Torehilin VP, Lukyanov AN, Gao Z, et al. Immunomieelles:targeted pharmaceutical carriers for poorly soluble drugs[J]. ProcNatl Acad Sci USA,2003, 100(10):6039-6044.
[8]Zeng J, Xu XY, Chen XS, Liang QZ, Bian XC, Yang LX, Jing XB. Biodegradable electrospun fibers for drug delivery. Journal of Controlled Release.2003,92: 227-311.
[9]李平祝,杨卓理,杨可伟,王菲,刘艳.多功能聚合物胶束的最新研究进展[J].中国新药杂志,2008,17(3)：199-202.
[10]J.S. Phang, W.A. Marsh, T.G Barlows 3rd, H.I. Schwartz. Determining feeding tube location by gastric and intestinal pH values. Nutr[J]. Clin. Pract.,2004,19: 64-644.
[11]M. Stubbs, P.M. McSheehy, J.R. Griffiths. Causes and consequences of acidic pH in rumors:a magnetic resonance study[J]. Adv. Enzyme. Regul.,1999,39:13-30.
[12]M. Dellian, G Helmlinger, E Yuan. R. K Jain. Fluorescence ratio imaging of
[12]M. Dellian, G Helmlinger, E Yuan. R. K Jain. Fluorescence ratio imaging of interstitial pH in solid turnouts:effect of glucose on spatial and temporal gradients[J]. Br. J. Cancer.,1996,74:1206-1215.
[13]L. Luo, J. Tam, D. Maysinger. Cellular Internalization of Poly(ethylene oxide)-b-poly(t-caprolactone)Dibiock Copolymer Micelles. Bioconjugate. Chem. 2002,13(6):1259-1265.
[14]I. Mellman, R. Fuchs, A. Helenius. Acidification of the endocytic and exocytic Pathways. Annu. Rev. Biochem.1986,55(4):773-700.
[15]Na K, Lee S, Bae YH. Polymeric micelle for tumor pH and folate-mediated targeting[J]. Control Release,2003,91(1/2):103-113.
[16]E.R, Gillies, J. M. Frechet. Structural isomerism in CuSCN coordination Polymers. Chem. Commun.2002.15:1640-1640.
[17]Rijcken CJ, Veldhuts TF, Ramzi A, et al. Novel fast degradable thermosensitiv polymerle micelles based on PEG-block-poly(N-(2-hydroxyethyl) methacrylamide-oligolactates) [J]. Biomacromolecules,2005,6(4):2343-2351.
[18]Lo CL, Huang CK, Lin KM, et al, Mixed micelles formed from graft and diblock copolymets for application in intracellular drug delivery [J]. Biomaterials, 2007,28(6):1225-1235.
[19]Lowe A B, Billingham N C, Armes S P, Synthesis and characterization of zwitterionic block copolymers, Macromolecules,1998,31(18):5991-5998.
[20]Tomalia DA, BakerH. Dewdd J, et al.A new class of polymers: starburst-dendritic macromolecules[J]. Polymer J,1985,17:117-132.
[21]Roberts. J C, BhMgat M, Zera R T. Preliminary biological evaluation of polyamidoamine (PAMAM)starburst dendrimers[J]. Biomed Matter Res.,1996, 30:53-65.
[22]Malik N, wiwarranapatapee R, Klopsch P, et al, Dendrimers:Relationship between structure and biocompatibility in vitro and preliminary studies on the biodistribution ofI251-labled polyamidoamine dendrimers in vivo[J]. Controlled Release,2000,65:133-148.
[23]Haensler J, Szoka F C Jr. Polyamidoamine cascade polymers mediate eficient transfection of cells in culture[J]. Bioconjugate Chem.,1993,4:372-379.
[24]Bielinska A, Kukowska L J F, Johnson J, et al. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendfimers[J]. Nucleic Acids Res,1996, 24:2176-2182.
[25]Zhao R X, Du B, Lu Z R, In vitro release of5-fluorouracil with cyclic core dendritic polymer[J], Cont wiled Release,1999,57:249-257.
[26]肖娟,陆华中,邹萍.树枝状聚合物在生物医学领域的应用进展[J].中国生物工程杂志,2002,22(4)：6-11.
[27]Esfand R, Tomalia Da. Poly(amidoamine)(PAMAM)dend rimers:from biomimicry to drug delivery and biomedical applications[J]. Drug Discov Today, 2001,6(8):427-436.
[28]Wooley K L, Hawker C J, Frechet J M J. Hyper-branched macromolecules via a novel double-stage convergent growth approach[J]. Journal of the American Chemical Society,1991,113(11):4252-4261.
[29]Spindler R, Frechet J M J. Two-step approach to-wards the accelerated synthesis of dendritic macromolecules [J]. Journal of the American Chemical Society: PerkinTransactions,1993,1:913-918.
[30]Sadler. K., Tam. J.P., Peptide dendrimers:applications and synthesis. Biotechnol, 2002,90(3-4):195-229.
[31]Bell T W, Science,1996 (271):1077-1078.
[32]沙耀武,武劫.树枝状大分子的研究进展与应用前景[]].湖南化工('Hunan Chemical Industry),2000,30 (4):1-5.
[33]D K Smith, Tetrahedron, Recent developments in dendrimer chemistry[J].An introduction.2003,59:3797-798.
[34]Chow H F, Mong T K K, Nongrum M F, et al. The synthesis and properties of novel functional dendritic molecules. Tetrahedron,1998,54:8543-8660.
[35]Cloninger C R.Biological applications of dendrimers. Curr Opin Chem Biol, 2002,6:742-748.
[36]Bodanzky M. Principles of Peptide Synthesis. Springer:New York 1984.
[37]Sakakibara S. Synthesis of large peptides in solution [J]. Biopolymers.1999(51): 279-296.
[38]Li X F, Zhang L, Zhang W. Solid-Phase Synthesis of 1,2,3,4-Tetrahydro-p-carboline-containing Peptidomimetics.Org Lett,2000(2):3075-3078.
[39]Kates S A. Albericio F. Solid-Phase Synthesis. A Practical Guide. Marcel Dekker: New York,2000:1-77.
[40]Merrifield R B. J Am Chem Soc,1963,85:2149-2156.
[41]Luo Y, Prestwich G D. Cancer-targeted polymeric drugs [J]. Curr Cancer Drug Targets,2002,2:209-226.
[42]Bhadra D, Bhadra S, Jain S, Jain N K. International Journal of Pharmaceutics, 2003,257:111-124.
[43]Ong K K, Jenkins A L, Cheng R, Tomalia D A, Durst H D, Jensen J L, Emanuel P A, Swim C R, Yin R. Analytica Chimica Acta,2001,444:143-148.
[44]Naylor, A. M. et al. Starburst dendrimers. Molecular shape control[J]. J. Am. Chem. Soc,1989,111:2339-2341.
[45]Jansen, J. F. G. A., de Brabander Vall den Berg, E. M. M. Meijer, E. W. Encapsulation of guest molecules into a dendritic box[J]. Science 1994 266:1226-1229.
[46]Watkins, D. M, et al. Dendrimers with hydrophobic cores and the formation of supramolecular dendrimer-surfactant assemblies[J]. Langmuir,1997,13:3136-3141.
[47]Newkome G R, Y Z Qi, Baker G R, et al. Cascade molecules:Synthesis and characterization of a benzene[9]3-arborol[J]. Journal of the American Chemical Society.1986,108(4):849-850.
[48]Stevelmann S, Van Hest J C M, Jansen J F G A,et al. Synthesis, characterization, and guest-host properties of inverted unimolecular dendritic micelles [J]. Journal of the American Chemical Society,1996,118(31):7398-7399.
[49]Hawker C J, Wooley K L, FrechetJ M J. Unimolecular micelles and globular amphiphiles:Dendritic macromolecules as novel recyclable solubilization agentsf [J]. Journal of the American Chemical Society:Perkin Transactions,1993,1:1287-1297.
[51]Kojima C, Kono K, Maruyama K, et al. Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate arlticancer drugs[J]. Bioconjugate Chemistry,2000,11(6):910-917.
[52]Gitsov I, Frechet J M J. Solution and solid-state properties of hybrid linear-dendritic block copolymers[J]. Macromolecules,1993,26(24):6536-6546.
[53]L Y Zhu, G L Zhu, M Z Li, et al. Thermosensitive aggregates self-assembled by an asymmetric block copolymer of dendritic polyether and poly (N-isopro pylacry-lamide) [J]. European Polymer Journal,2002,38(12):2503-2506.
[54]Beezer A E, King A S H, Martin I K, et al. Dendrimers as potential drug carriers: Encapsulation of acidic hydrophobes within water soluble PAMAM derivatives [J]. Tetrahedron,2003,59(22):3873-3881.
[55]L Y Jie, Dubin P L, Spindler R, et al. Complexformation between poly(dimethyldiallylammonium chloride)and carboxylated starburst dendrimers [J]. Macromolecules,1995,28(24):8426-8428.
[56]陈双玲,叶玲.树状大分子：一类新型的纳米容器和输送装置[J].国外医学：药学分册,2004,31(3)：181-184.
[57]Yang H, Lopina S T. Penicillin V-conjugated PEG-PAMAM star polymers [J]. Journal of Bio materials Scienee:Polymer Edition,2003,14(10):1043-1056.
[2]张大为.功能化高分子胶束药物载体的制备与研究[学].北京化工大学,2010.
[3]Amedo A, lrache J M, Merodio M, et al. AlbuIllin nanopmiclesimproved the stability, nuclear accumulation and anticytomegaloviral activity of aphosphodiester oligonucleotide[J]. Controlled Release,2004,94(1):217.
[4]可伟,杨卓理,孙玉峰,刘艳.大分子胶束作为药物载体的应用[J].中国新药杂志,2006,15(19)：1630-1635.
[5]Allen C, Maysinger D, EisenbcIg A. Nano. engineering block copolymer aggregates for drug delivery[J]. colloids SurfB,1999,16:1-35.
[6]Yu B G, Okano T, Kataoka K, et al. Polymeric micelle for drug delivery; solubilizafion and haemoiytie activity of amphotericin B[J]. J Controlled Release, 1998,53(1-3):131-136.
[7]Torehilin VP, Lukyanov AN, Gao Z, et al. Immunomieelles:targeted pharmaceutical carriers for poorly soluble drugs[J]. ProcNatl Acad Sci USA,2003, 100(10):6039-6044.
[8]Zeng J, Xu XY, Chen XS, Liang QZ, Bian XC, Yang LX, Jing XB. Biodegradable electrospun fibers for drug delivery. Journal of Controlled Release.2003,92: 227-311.
[9]李平祝,杨卓理,杨可伟,王菲,刘艳.多功能聚合物胶束的最新研究进展[J].中国新药杂志,2008,17(3)：199-202.
[10]J.S. Phang, W.A. Marsh, T.G Barlows 3rd, H.I. Schwartz. Determining feeding tube location by gastric and intestinal pH values. Nutr[J]. Clin. Pract.,2004,19: 64-644.
[11]M. Stubbs, P.M. McSheehy, J.R. Griffiths. Causes and consequences of acidic pH in rumors:a magnetic resonance study[J]. Adv. Enzyme. Regul.,1999,39:13-30.
[12]M. Dellian, G Helmlinger, E Yuan. R. K Jain. Fluorescence ratio imaging of
[12]M. Dellian, G Helmlinger, E Yuan. R. K Jain. Fluorescence ratio imaging of interstitial pH in solid turnouts:effect of glucose on spatial and temporal gradients[J]. Br. J. Cancer.,1996,74:1206-1215.
[13]L. Luo, J. Tam, D. Maysinger. Cellular Internalization of Poly(ethylene oxide)-b-poly(t-caprolactone)Dibiock Copolymer Micelles. Bioconjugate. Chem. 2002,13(6):1259-1265.
[14]I. Mellman, R. Fuchs, A. Helenius. Acidification of the endocytic and exocytic Pathways. Annu. Rev. Biochem.1986,55(4):773-700.
[15]Na K, Lee S, Bae YH. Polymeric micelle for tumor pH and folate-mediated targeting[J]. Control Release,2003,91(1/2):103-113.
[16]E.R, Gillies, J. M. Frechet. Structural isomerism in CuSCN coordination Polymers. Chem. Commun.2002.15:1640-1640.
[17]Rijcken CJ, Veldhuts TF, Ramzi A, et al. Novel fast degradable thermosensitiv polymerle micelles based on PEG-block-poly(N-(2-hydroxyethyl) methacrylamide-oligolactates) [J]. Biomacromolecules,2005,6(4):2343-2351.
[18]Lo CL, Huang CK, Lin KM, et al, Mixed micelles formed from graft and diblock copolymets for application in intracellular drug delivery [J]. Biomaterials, 2007,28(6):1225-1235.
[19]Lowe A B, Billingham N C, Armes S P, Synthesis and characterization of zwitterionic block copolymers, Macromolecules,1998,31(18):5991-5998.
[20]Tomalia DA, BakerH. Dewdd J, et al.A new class of polymers: starburst-dendritic macromolecules[J]. Polymer J,1985,17:117-132.
[21]Roberts. J C, BhMgat M, Zera R T. Preliminary biological evaluation of polyamidoamine (PAMAM)starburst dendrimers[J]. Biomed Matter Res.,1996, 30:53-65.
[22]Malik N, wiwarranapatapee R, Klopsch P, et al, Dendrimers:Relationship between structure and biocompatibility in vitro and preliminary studies on the biodistribution ofI251-labled polyamidoamine dendrimers in vivo[J]. Controlled Release,2000,65:133-148.
[23]Haensler J, Szoka F C Jr. Polyamidoamine cascade polymers mediate eficient transfection of cells in culture[J]. Bioconjugate Chem.,1993,4:372-379.
[24]Bielinska A, Kukowska L J F, Johnson J, et al. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendfimers[J]. Nucleic Acids Res,1996, 24:2176-2182.
[25]Zhao R X, Du B, Lu Z R, In vitro release of5-fluorouracil with cyclic core dendritic polymer[J], Cont wiled Release,1999,57:249-257.
[26]肖娟,陆华中,邹萍.树枝状聚合物在生物医学领域的应用进展[J].中国生物工程杂志,2002,22(4)：6-11.
[27]Esfand R, Tomalia Da. Poly(amidoamine)(PAMAM)dend rimers:from biomimicry to drug delivery and biomedical applications[J]. Drug Discov Today, 2001,6(8):427-436.
[28]Wooley K L, Hawker C J, Frechet J M J. Hyper-branched macromolecules via a novel double-stage convergent growth approach[J]. Journal of the American Chemical Society,1991,113(11):4252-4261.
[29]Spindler R, Frechet J M J. Two-step approach to-wards the accelerated synthesis of dendritic macromolecules [J]. Journal of the American Chemical Society: PerkinTransactions,1993,1:913-918.
[30]Sadler. K., Tam. J.P., Peptide dendrimers:applications and synthesis. Biotechnol, 2002,90(3-4):195-229.
[31]Bell T W, Science,1996 (271):1077-1078.
[32]沙耀武,武劫.树枝状大分子的研究进展与应用前景[]].湖南化工('Hunan Chemical Industry),2000,30 (4):1-5.
[33]D K Smith, Tetrahedron, Recent developments in dendrimer chemistry[J].An introduction.2003,59:3797-798.
[34]Chow H F, Mong T K K, Nongrum M F, et al. The synthesis and properties of novel functional dendritic molecules. Tetrahedron,1998,54:8543-8660.
[35]Cloninger C R.Biological applications of dendrimers. Curr Opin Chem Biol, 2002,6:742-748.
[36]Bodanzky M. Principles of Peptide Synthesis. Springer:New York 1984.
[37]Sakakibara S. Synthesis of large peptides in solution [J]. Biopolymers.1999(51): 279-296.
[38]Li X F, Zhang L, Zhang W. Solid-Phase Synthesis of 1,2,3,4-Tetrahydro-p-carboline-containing Peptidomimetics.Org Lett,2000(2):3075-3078.
[39]Kates S A. Albericio F. Solid-Phase Synthesis. A Practical Guide. Marcel Dekker: New York,2000:1-77.
[40]Merrifield R B. J Am Chem Soc,1963,85:2149-2156.
[41]Luo Y, Prestwich G D. Cancer-targeted polymeric drugs [J]. Curr Cancer Drug Targets,2002,2:209-226.
[42]Bhadra D, Bhadra S, Jain S, Jain N K. International Journal of Pharmaceutics, 2003,257:111-124.
[43]Ong K K, Jenkins A L, Cheng R, Tomalia D A, Durst H D, Jensen J L, Emanuel P A, Swim C R, Yin R. Analytica Chimica Acta,2001,444:143-148.
[44]Naylor, A. M. et al. Starburst dendrimers. Molecular shape control[J]. J. Am. Chem. Soc,1989,111:2339-2341.
[45]Jansen, J. F. G. A., de Brabander Vall den Berg, E. M. M. Meijer, E. W. Encapsulation of guest molecules into a dendritic box[J]. Science 1994 266:1226-1229.
[46]Watkins, D. M, et al. Dendrimers with hydrophobic cores and the formation of supramolecular dendrimer-surfactant assemblies[J]. Langmuir,1997,13:3136-3141.
[47]Newkome G R, Y Z Qi, Baker G R, et al. Cascade molecules:Synthesis and characterization of a benzene[9]3-arborol[J]. Journal of the American Chemical Society.1986,108(4):849-850.
[48]Stevelmann S, Van Hest J C M, Jansen J F G A,et al. Synthesis, characterization, and guest-host properties of inverted unimolecular dendritic micelles [J]. Journal of the American Chemical Society,1996,118(31):7398-7399.
[49]Hawker C J, Wooley K L, FrechetJ M J. Unimolecular micelles and globular amphiphiles:Dendritic macromolecules as novel recyclable solubilization agentsf [J]. Journal of the American Chemical Society:Perkin Transactions,1993,1:1287-1297.
[51]Kojima C, Kono K, Maruyama K, et al. Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate arlticancer drugs[J]. Bioconjugate Chemistry,2000,11(6):910-917.
[52]Gitsov I, Frechet J M J. Solution and solid-state properties of hybrid linear-dendritic block copolymers[J]. Macromolecules,1993,26(24):6536-6546.
[53]L Y Zhu, G L Zhu, M Z Li, et al. Thermosensitive aggregates self-assembled by an asymmetric block copolymer of dendritic polyether and poly (N-isopro pylacry-lamide) [J]. European Polymer Journal,2002,38(12):2503-2506.
[54]Beezer A E, King A S H, Martin I K, et al. Dendrimers as potential drug carriers: Encapsulation of acidic hydrophobes within water soluble PAMAM derivatives [J]. Tetrahedron,2003,59(22):3873-3881.
[55]L Y Jie, Dubin P L, Spindler R, et al. Complexformation between poly(dimethyldiallylammonium chloride)and carboxylated starburst dendrimers [J]. Macromolecules,1995,28(24):8426-8428.
[56]陈双玲,叶玲.树状大分子：一类新型的纳米容器和输送装置[J].国外医学：药学分册,2004,31(3)：181-184.
[57]Yang H, Lopina S T. Penicillin V-conjugated PEG-PAMAM star polymers [J]. Journal of Bio materials Scienee:Polymer Edition,2003,14(10):1043-1056.