新型生物材料聚β苹果酸β羟基丁酸酯共聚物的合成及其性质研究论文.docx

1、新型生物材料聚苹果酸羟基丁酸酯共聚物的合成及其性质研究论文新型生物材料聚-苹果酸/-羟基丁酸酯共聚物的合成及其性质研究 目 录缩略语表 1中文摘要 3Abstract 5前 言 8文献回顾 101天然药用高分子材料 101.1多糖类 101.2蛋白质类 111.3多肽类 121.3 聚酯类和其他 132脂肪族聚酯类的研究及应用 142.1 -聚苹果酸的研究及应用 142.1.1 -聚苹果酸及其共聚物的合成研究 152.1.2 -聚苹果酸及其共聚物在药物载体方面的应用 162.2聚-羟基丁酸酯的研究及应用 172.2.1 聚-羟基丁酸酯的性质及其改性研究 172.2.2 聚-羟基丁酸酯及其衍生物

2、在药物物载体方面的应用 183 课题的提出 19正 文 21第一部分 聚-羟基丁酸酯和共聚物的合成 211 材料与仪器 211.1实验试剂 211.2 实验仪器 222 实验方法 232.1聚-羟基丁酸酯的合成 232.2共聚物的合成 232.2.1 -苹果酸苄基内酯单体的合成及密度测定 232.2.2 P(MLABe-co-BL)和PMLABe-co-PHB的合成 242.2.3 P(MLA-co-BL)和PMLA-co-PHB的合成 252.3测试与表征 263 结果与讨论 263.1聚-羟基丁酸酯的合成 263.2 -苹果酸苄基内酯单体的合成 293.3 P(MLABe-co-BL) 和

3、PMLABe-co-PHB的合成 303.3微观聚合机理分析 343.4 P(MLA-co-BL)的合成 37第二部分 共聚物的性质研究 391 材料与仪器 391.1 实验试剂 391.2 实验仪器 392 实验方法 402.1共聚物粒径、zeta电位和溶解度的测定 402.1.1粒径测定 402.1.2 Zeta电位测定 402.1.3溶解度测定 402.2共聚物的降解 402.2.1 P(MLABe-co-BL)的降解 402.2.2 P(MLA-co-BL)和PMLA-co-PHB的降解 412.3 细胞毒性测定 412.3.1细胞的培养 412.3.2 P(MLABe-co-BL)的

4、细胞毒性实验 412.3.3 P(MLA-co-BL) 和PMLA-co-PHB的细胞毒性实验 422.4 溶血实验 422.4.1 兔全血的采集和2 %红细胞混悬液的配制 422.4.2 P(MLABe-co-BL)的溶血实验 432.4.3 P(MLA-co-BL) 和PMLA-co-PHB的溶血实验 433 结果与讨论 433.1粒径、zeta电位和溶解度的测定 433.2共聚物的降解 443.2.1 P(MLABe-co-BL)的降解 443.2.2 P(MLA-co-BL) 和PMLA-co-PHB 的降解 463.3细胞毒性测定 473.3.1 P(MLABe-co-BL)的细胞毒

5、性测定 473.3.2 P(MLA-co-BL) 和PMLA-co-PHB的细胞毒性测定 473.4溶血实验 483.4.1 P(MLABe-co-BL)的溶血实验 483.4.2 P(MLA-co-BL) 和PMLA-co-PHB的溶血实验 48第三部分 PMLA-co-PHB的应用和展望 501 材料与仪器 501.1 实验材料 501.2 实验仪器 502 实验方法 512.1 PMLA-co-PHB空白胶束的制备 512.2 PMLA-co-PHB包封色胺酮胶束的制备 522.3 PMLA-co-PHB胶束包封率和载药率的测定 522.4 PMLA-co-PHB临界胶束浓度（CMC）的

6、测定 533 结果与讨论 533.1 PMLA-co-PHB胶束的制备 534 材料的应用展望 544.1 在药物载体方面的应用 554.2在其它方面的应用 55小 结 57参考文献 58附 录 67研究成果 71致 谢 72缩略语表缩略词英文全称中文全称BL-butyrolactone -丁内酯MLABebenzyl-malolactone-苹果酸苄基内酯TFAATrifluoroaceticanhydride三氟乙酸酐P(MLABe-co-BL)Poly(benzyl-malolactone-co-butyrolactone)聚（-苹果酸苄基酯-co-羟基丁酸酯）P(MLA-co-BL)P

7、oly (-malic acid-co-butyrolactone) 聚（-苹果酸-co-羟基丁酸酯）IRInfrared Spectroscopy红外光谱CMCCritical Micelle Concentration临界胶束浓度GPCGel Permeation Chromatography凝胶渗透色谱PHBPoly (-hydroxybutyrate)聚-羟基丁酸酯PMLAPoly (-malic acid)-聚苹果酸MwWeight-average Molecular Weight重均分子量PMLABe-co- PHBPoly(benzyl-malolactone)-co-Poly

8、(-hydroxybutyrate)聚-苹果酸苄基酯-co-聚羟基丁酸酯PMLA-co- PHBPoly(-malic acid)-co-Poly (-hydroxybutyrate)-聚苹果酸-co-聚羟基丁酸酯PHAspolyhydroxyalkanoates聚羟基脂肪酸MnNumber-average Molecular Weight数均分子量PCLpolycaprolactone聚己内酯NMRNuclear Magnetic Resonance核磁共振PBSPhosphate Buffered Saline磷酸盐缓冲液THFTetrahydrofuran四氢呋喃PEOpolyoxyet

9、hylene聚氧化乙烯PVAPoly(vinylalcohol)聚乙烯醇PLAPoly(lactic acid)聚乳酸PDIPolydispersity Index多分散系数PLGAPoly(lactide-co-glycolide)聚（丙交酯-co-乙交酯）ROPRing-opening Polymerization开环聚合法PASPPolysucciuimide聚天冬氨酸Y-PGAPoly glutamic acid聚谷氨酸PLHPoly-L-histidine聚组氨酸PArgPoly-L-arginine聚精氨酸PLPolylysine聚赖氨酸GSHGlutathione谷胱甘肽PEGP

10、olyethylene glycol聚乙二醇MDIMethylenediphenyl Diisocyanate二苯基甲烷二异氰酸酯新型生物材料聚-苹果酸/-羟基丁酸酯共聚物的合成及其性质研究中文摘要聚苹果酸（PMLA）是一种结构规整的水溶性脂肪族聚酯，具有生物降解性、生物相容性和无免疫原性，主链上具有多个悬挂羧基，可以很好的被修饰或改性形成聚合物前药，但PMLA亲水性强、降解过快，用作生物高分子材料存在诸多缺陷。聚-羟基丁酸酯（PHB）是一种由细菌发酵产生的热塑性聚酯，具有良好的生物相容性，但存在易结晶、疏水性强和降解时间长等缺点。本课题拟采用PHB改性PMLA，以期达到优势互补，改善聚苹果酸

11、的性能。以苯甲酸四乙铵为引发剂，通过阴离子开环引发-苹果酸苄基内酯（MLABe）和-丁内酯（BL）开环共聚。研究中考察了不同比例的混合单体在不同的反应时长、反应温度及不同的加入顺序等条件对共聚的影响。两单体通过混合均匀后同时聚合或是分时段加入而分别得到两种疏水性的共聚物：无规共聚物聚（-苹果酸苄基酯-co-羟基丁酸酯）（P(MLABe-co-BL)）和嵌段共聚物-聚苹果酸苄基酯-co-聚羟基丁酸酯（PMLABe-co-PHB）。氢化苄基后分别得到两亲性的共聚物：聚（-苹果酸-co-羟基丁酸酯）（P(MLA-co-BL)）和-聚苹果酸-co-聚羟基丁酸酯（PMLA-co-PHB）。在共聚之前，先

12、优化了-苹果酸苄基内酯单体的合成过程。课题组前期的研究已表明引发剂苯甲酸四乙铵能够引发-苹果酸苄基内酯单体开环聚合形成PMLA，但未证实对-丁内酯有同样的引发作用，本实验用1H-NMR， 13C-NMR和 FT-IR表征了-丁内酯开环聚合形成的产物聚-羟基丁酸酯，证实该引发剂对-丁内酯有同样的开环作用，为-苹果酸苄基内酯和-丁内酯单体共聚提供实验基础。对无规共聚物P(MLABe-co-BL)聚合过程的研究发现，加入不同聚合单体的比例，共聚物组成有明显差别。MLABe的竞聚率比BL的大，加入BL的比率越多，聚合时间就越长。MLABe/BL为50/50（mol/mol）时的微观聚合机理表明，聚合过

13、程中酯交换剧烈，趋向于无规共聚。MLABe/BL为 75/25（mol/mol）时，聚合时间短、共聚产物分子量适中且分布宽度窄，所以后续实验均使用该比例聚合。P(MLA-co-BL)、PMLA-co-PHB的亲水性、降解性和溶解度分别介于P(MLABe-co-BL)和PMLA之间，改善了PMLA用做药物载体降解快、负电性大的特点。通过细胞毒性实验和溶血实验发现P(MLABe-co-BL)有一定的细胞毒性，但没有溶血现象。低浓度（0.4 mg/mL以下）的P(MLA-co-BL)没有抑制细胞增长，反而具有促进作用；随着浓度的增加，在高于0.8 mg/mL后有轻微的抑制作用。嵌段共聚物PMLA-c

14、o-PHB 随着浓度的增加没有呈现出细胞毒性。在溶血实验中，材料浓度为 0.2 mg/mL 时，无规共聚物的溶血率（0.5543 0.05 %）小于嵌段共聚物（3.249 0.04 %），可能是嵌段共聚物相比无规共聚物在溶液中更容易形成胶束。随着材料浓度的增加，细胞成棕褐色并有沉降产生。课题进一步开展了嵌段共聚物PMLA-co-PHB形成纳米胶束用作药物载体的研究。以色胺酮为药物模型通过透析法制备载药胶束，包封率为8.35 0.29 %，载药率为2.67 0.2 %，粒径为242 nm。为PMLA-co-PHB用作聚合物纳米药物运载体系提供了前期实验基础。下一步我们将开展PMLA-co-PHB

15、作为药物载体以及P(MLA-co-BL)在组织工程支架方面的应用。关键词：-苹果酸苄基内酯；-羟基丁酸酯；聚合物胶束；聚合物药物载体；降解Synthesis and biological properties of new biological materials -malic acid/-butyrolactone copolymersAbstractPoly(-L-malic acid) (PMLA) is a natural aliphatic polyester, and it was proved to be biodegradable, non-toxic and non-immu

16、nogenic. Furthmore, it could provide multiple suspend carboxyl to form polymer pro-drugs. However, its strong hydrophilicity and fast degradation time limits its application. Poly(-hydroxybutyrate) (PHB) is a thermoplastic polyester produced by a bacterial fermentation, and it has good biocompatibil

17、ity. But it is strongly hydrophobic and shows long degradation time. In this study, PHB was used to modify PMLA, realizing complementary advantages.The copolymer was synthesized byring-openingpolymerization ofbenzyl-malo- lactone (MLABe)and-butyrolactone (BL), using tetraethylammonium benzoic acidas

18、 initiator. Ratio of the two monomers, reaction times, temperatures and adding orders were studied to examine the impact of these factors on the copolymerization. Two monomers were added simultaneously or successively, and two different hydrophobic polymers were obtained: random copolymers P(MLABe-c

19、o-BL) and block copolymers PMLABe-co-PHB. Amphiphilic copolymers P(MLA-co-BL) and PMLA-co-PHB were obtained by hydrogenation. The synthetic process of benzyl-malolactone monomers was optimized before the copolymerization. In our previous study, the initiator tetraethylammonium benzoate was used to t

20、rigger the ring-opening polymerization to form PMLA. However, this study still need to confirm if the initiator can also trigger the polymerization of -butyrolactone. 1H-NMR, 13C-NMR and FT-IR were used to characterize the polymer poly(-hydroxybutyrate) (PHB). The results showed that the initiator c

21、ould be used to initiate the polymerization of -butyrolactone, which laid the foundation for copolymerization reaction.Research on polymerization process of the random copolymer P(MLABe-co-BL) showed that there is a significant differences in the composition of the copolymers when different ratio of

22、 monomers added. Scale of micro polymerization mechanism indicated that the ratio of MLABeand BL is 50/50 (mol/mol), which result in severe transesterification in the polymerization process, tends to random copolymerization. When the ratio is 75/25 (mol/mol), copolymerization products obtained in sh

23、ort time which have moderate molecular weight and narrow distribution width. Therefore, all of the subsequent polymerization experiments take this ration. The hydrophilicity, degradability and solubility of P(MLA-co-BL) and PMLA-co-PHB were between that of P(MLABe-co-BL) and PMLA, which could overco

24、me the shortcomings of PMLA, such as fast degradation and stongly negative charge. In vitro cytotoxicity study found that P(MLABe-co-BL) showed certain cytotoxicity. The result of hemolysis assay suggested that P(MLABe-co-BL) possess good biocompatibility. P(MLA-co-BL) did not inhibit cell growth at

25、 low concentrations (0.4 mg/mL or less). A slight inhibition would happen with increasing concentration to 0.8 mg/mL. Accordingly, the block copolymers PMLA-co-PHB with increasing concentration did not exhibit cytotoxicity. In hemolysis test, the concentration of material at 0.2 mg/mL, hemolysis rat

26、e of random copolymer is 0.5543 0.05 %, less than that of block copolymer, 3.249 0.04 %. This suggested that random copolymer may be easier to form micelles in aqueous solution compared to block copolymer. With the increasing of concentration of material, cells turned into brown and settled onto the

27、 bottom.The research of nanomicelles formed by PMLA-co-PHB block copolymer used as drug carriers was further studied in this subject. Tryptanthrin severed as a drug model, drug loaded nanosphere was prepared by dialysis method. Entrapment efficiency is 8.35 0.29 %, drug-loaded rate is 2.67 0.2 %, an

28、d particle size is 242 nm. The results provided a preliminary experimental basis that PMLA-co-PHB used as Polymer nano drug delivery system. The next step in this work is to develop PMLA-co-PHB as drug carriers, and exploit the application of P(MLA-co-BL) in tissue engineering scaffolds.Keyword: ben

29、zyl-malolactone; -hydroxybutyrate; polymeric micelles; polymer drug carrier; degradation 前 言 聚苹果酸（PMLA）是一类新型生物材料，具有生物可降解性、生物相容性、无免疫原性等特点。在水溶液中PMLA主链上的酯键可发生水解或酶促降解，从而生成无毒无害的小分子苹果酸，并参与体内三羧酸循环而被生物体吸收1。聚苹果酸结构规整，具有多个悬挂羧基，可以很好的被修饰或改性形成聚合物前药，应用于药物缓释和组织工程等领域2。但大量羧基的存在使得PMLA亲水性强、降解过快3，用作生物高分子材料存在诸多缺陷，因此，对其进行

30、疏水性修饰，在一定程度上降低其水溶性就显得非常有必要。聚-羟基丁酸酯（PHB）是一种由原核微生物发酵产生的热塑性聚酯，具有良好生物相容性4，但存在易结晶、疏水性强和降解时间长5等缺点。本课题采用PHB改性PMLA，以-苹果酸苄基内酯（MLABe）和-丁内酯（BL）开环共聚达到改性的目的。通过化学方法共聚合成聚苹果酸衍生物很少有报道，除了与聚丙内酯，聚己内酯和聚丙交酯外6，目前仅Guillaume课题组用有机和金属有机催化剂引发-苹果酸苄基内酯和-丁内酯共聚，但金属引发剂和有机催化剂都存在一定的毒性残留7。本实验以阴离子引发剂引发二者开环共聚。在共聚之前，先优化了-苹果酸苄基内酯单体的合成过程。

31、课题组前期的研究已表明引发剂苯甲酸四乙铵能够引发-苹果酸苄基内酯单体开环聚合形成PMLA，但未证实对-丁内酯有同样的引发作用，为-苹果酸苄基内酯和-丁内酯单体能够开环共聚提供实验基础。以阴离子开环的机理引发MLABe和BL开环共聚得到一定分子量的疏水性共聚物P(MLABe-co-BL)和PMLABe-co-PHB，二者氢化苄基后分别得到两亲性的共聚物P(MLA-co-BL)和PMLA-co-PHB。得到的共聚物是生物相容性和生物降解性均良好的两亲性聚羟基脂肪酸酯（PHAs），为其在药物载体、组织工程支架等后续生物医学研究方面奠定了实验基础。1999年Cammas,S.等合成聚羟基脂肪酸酯，其在体内具有良好的生物相容性、水溶性和生物降解性，在氧气充分的情况下，通过三羧酸循环最终降解为水和二氧化碳等小分子无毒物质排出体外2，其在药物缓释体系