基于响应性聚合物组装体的生物医用与检测功能材料.pdf

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1、 中国科学技术大学 博士学位论文 基于响应性聚合物组装体的 生物医用与检测功能材料 基于响应性聚合物组装体的 生物医用与检测功能材料 作者姓名：万学娟 学科专业：高分子化学与物理 导师姓名：刘世勇 教授 完成时间：二一一年四月二十九日 University of Science and Technology of China A dissertation for doctors degree Responsive Polymeric Assemblies-Based Functional Materials for Biomedical and Detection Applications A

2、uthors Name：Xuejuan Wan Speciality：Polymer Chemistry and Physics Supervisor：Prof.Shiyong Liu Finished time:Apr 29th,2011 中国科学技术大学学位论文原创性声明 本人声明所呈交的学位论文,是本人在导师指导下进行研究工作所取得的成果。除已特别加以标注和致谢的地方外，论文中不包含任何他人已经发表或撰写过的研究成果。与我一同工作的同志对本研究所做的贡献均已在论文中作了明确的说明。作者签名：_ 签字日期：_ 中国科学技术大学学位论文授权使用声明 作为申请学位的条件之一，学位论文著作权拥有

3、者授权中国科学技术大学拥有学位论文的部分使用权，即：学校有权按有关规定向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅，可以将学位论文编入中国学位论文全文数据库等有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存、汇编学位论文。本人提交的电子文档的内容和纸质论文的内容相一致。保密的学位论文在解密后也遵守此规定。公开 保密（_年）作者签名：_ 导师签名：_ 签字日期：_ 签字日期：_ 摘 要 I摘摘 要要 在过去十年中，响应性聚合物自组装方面的相关研究已相当深入和系统。响应性聚合物超分子自组装体在催化化学、材料制备、生物医药等多方面具有极为广泛的应用价值，已经成为 21

4、 世纪最重要的研究课题之一。目前该领域未来的发展方向应该是在现有的研究成果基础上，利用响应性聚合物的独特性质探索和发展新型功能材料和器件。本论文结合活性自由基聚合，点击化学与生物偶联等方法制备了多种具有不同组成，不同拓扑结构的响应性聚合物，详细研究了这些聚合物的组装行为，并在此基础之上，进一步将响应性聚合物与无机纳米技术，化学传感概念相结合，发展了新型的智能药物传输载体以及更灵敏的比率型化学传感体系。具体来说，本论文的工作包括以下四个方面：1.结合原子转移自由基聚合(ATRP)，开环聚合(ROP)和点击化学合成了两亲性热敏感环-线形二嵌段聚合物环形聚(N-异丙基丙烯酰胺)-b-线形聚己内酯(c

5、-PNIPAM)-b-PCL。首先，由特殊设计的含有炔基，羟基以及 ATRP 引发基元的三官能团分子 alkynyl-(OH)-Br 出发，用原子转移自由基聚合进行NIPAM 单体的聚合，并通过叠氮化反应和点击化学反应合成环形 PNIPAM前体(c-PNIPAM)-OH；接着利用(c-PNIPAM)-OH 为大分子引发剂，直接在 PNIPAM 环上进行开环聚合，得到结构明确的两亲性环-线嵌段共聚物(c-PNIPAM)-b-PCL。此外，为了对比，还合成了与目标环-线聚合物化学组成相类似的线型二嵌段聚合物(l-PNIPAM)-b-PCL，并详细比较了这两者在组装，以及药物负载/释放方面的性能差异

6、。2.结合 ATRP 与点击化学，将酶蛋白的辅助因子-锌配位原卟啉(PPIXZn)改性到温敏性 PNIPAM 长链的端基上，得到 PPIXZn-PNIPAM，然后将其与去除了原卟啉辅基的肌红蛋白(myoglobin)进行辅助因子重构反应，得到了生物杂化的热敏感双亲水性嵌段共聚物 myoglobin-b-PNIPAM。在较低的溶液温度下，所得嵌段共聚物单链溶解于水，而在较高的温度下，则会逐渐组装成具有生物活性，以疏水的 PNIPAM 为核，以亲水的肌红蛋白链为壳的胶束。另外，还结合 ATRP 和点击化学制备了有具有不同链构造和分子量的生物素(Biotin)功能化均聚物、二嵌段聚合物，其中生物素分

7、别被标记于共聚物与嵌段聚合物的链端或嵌段连接点处。利用生物素和亲和素(Avidin)之间强烈的特异性相互作用，可以有效的制备星型聚合物，星型二嵌段聚合物和杂臂星型聚合物等。高分子化的生物素由于聚合物链的存在，在与亲和素结合的过程表现出位阻效应。本节用 Avidin/HABA 分析法摘 要 II详细研究了这种位阻效应的规律。3.结合可控药物释放以及荧光检测两种功能，构筑了一种具有 pH 荧光检测功能的无机/有机杂化多孔硅药物可控释放体系。首先，在多孔硅的表面通过可逆加成-断裂链转移(RAFT)聚合原位共聚三种单体，分别是可交联因子聚(N-羟基琥珀酰亚胺丙烯酸酯)(NAS),生物相容的亲水性单体聚

8、乙二醇单甲醚甲基丙烯酸酯(OEGMA)，以及基于 1,8-萘酰亚胺的荧光 pH 检测单体(NaphMA)。所得的无机/有机杂化纳米粒子在水溶液中可以良好的分散，当环境 pH 在 4-8 之间的变化时也有明显的响应。在多孔硅中载入罗丹明 B(RhB)之后，可以利用胱胺交联多孔硅表面的聚合物刷。在还原剂二硫苏糖醇(DTT)存在条件下，多孔硅内包裹的 RhB 分子能够被释放出来。通过调节 DTT 的浓度，刺激释放的速率也可以得到有效地控制。此外，基于多孔硅材料还可实现多种药物共同释放。在另外一个体系中，我们利用 pH 可电荷反转的聚电解质多层膜P(DMA-co-TPAMA)/PAH来封堵装载了RhB

9、分子的多孔硅，并在聚电解质多层膜中负载抗癌药物顺铂。其中 PAH 是聚阳离子聚烯丙基胺，聚阴离子 P(DMA-co-TPAMA)是由亲水性的聚 N,N 二甲基丙烯酰胺(DMA)和pH可电荷反转的-羧基取代酰胺衍生物TPAMA经过自由基共聚而得。当环境 pH 值低于 6 时，聚阴离子中的-羧基取代酰胺键会断裂，产生电荷反转生成聚阳离子氨丙基甲基丙烯酰胺(APMA)。在静电斥力的作用下，多孔硅表面的聚电解质多层膜解离，从而释放多孔硅内包裹的 RhB 以及聚电解质层中吸附的顺铂，实现两种药物的共同释放。4.利用温度敏感的核交联胶束作为新型离子检测载体，实现对 Hg2+离子的比率型检测。首先，将 Hg

10、2+离子探针通过共聚的方式接入到温敏双亲水性嵌段聚合物 PEO-b-P(NIPAM-co-NAS-co-NUMA)中，并比较了此体系在不同温度，交联前后对 Hg2+离子的检测情况，其中 PEO，NUMA 分别为聚环氧乙烷和基于 1,8-萘酰亚胺的 Hg2+荧光检测单体。在 Hg2+离子存在条件下，检测体系的表观颜色会由黄色变为无色，荧光发射也会有相应的蓝移，由绿色荧光转变为蓝色荧光。将聚合物胶束交联后，体系对 Hg2+离子的检测限会进一步降低。同时，这种体系具有较好的生物相容性，在细胞中也可以灵敏的对 Hg2+离子进行检测。在另外一个体系中，我们还分别将荧光共振能量(FRET)给体 7-硝基-

11、2,1,3-苯并氧杂恶二唑(NBD)和两个 pH 敏感的 RhB-乙二胺衍生物受体分别标记到温敏性无规共聚物的中间以及两端，得到遥爪型聚合物 NBD-(P(OEGMA-co-DEGMA)-RhB2)。DEGMA 和OEGMA 分别是二甘醇单甲醚甲基丙烯酸酯和寡聚乙二醇单甲醚甲基丙烯酸酯。聚合物端基上的荧光受体 RhB-乙二胺衍生物经特殊设计，对环境的摘 要 IIIpH 值具有灵敏的响应，即在中性和碱性条件下没有荧光，而在酸性条件下出现强的荧光发射。此外，在较高温度下，无规共聚物 P(OEGMA-co-DEGMA)单链塌缩会拉近 NBD 与 RhB 之间的距离，增强 FRET 的效果，实现 pH

12、 和温度的双重比率型检测。关键词：关键词：可控/活性自由基聚合 点击化学 环境响应性 双亲水性嵌段共聚物 自组装 聚合物-蛋白质缀合物 多孔硅 药物控制释放 传感器 比率型探针 ABSTRACT IVABSTRACT Supramolecular assemblies fabricated from stimuli-responsive polymers have attracted considerable interests in the past decades due to their promising applications in diverse fields,such as c

13、atalysis,material preparation,and biomedicine,which render this interdisciplinary research subject as one of the promising scientific issues in the 21st century.The field of responsive polymers has nowadays been thoroughly and systematically explored,which evolved well beyond the demonstration of no

14、vel and interesting properties.Currently,the development of useful and advanced functions,e.g.,drug or gene carriers with triggered release properties,catalysis,detection and imaging,environmentally adaptive coatings,and self-healing materials,have emerged to be a more relevant subject.In this case,

15、we reported on the facile fabrication of numerous well-defined specific functionalized polymers with varying chemical architectures in the combination of controlled/living radical polymerization,click chemistry and bioconjugation protocols,and investigated their self-assembly behavior in aqueous sol

16、ution.Moreover,the following combination of stimuli-responsive polymers with inorganic nanoparticles and chemical sensors demonstrated that this kind of intelligent material can be utilized as promising functional nanocarriers for controlled drug delivery and ratiometric chemical sensing.Specificall

17、y,the dissertation includes the following four parts:1.We report a novel approach for the synthesis of amphiphilic and thermoresponsive tadpole-shaped linear-cyclic diblock copolymer,(c-PNIPAM)-b-PCL,consisting of hydrophobic linear poly(-caprolactone)(PCL)and thermoresponsive macrocyclic PNIPAM via

18、 the ring-opening polymerization(ROP)of CL monomer directly initiating from the cyclic PNIPAM precursor bearing a single hydroxyl functionality.We then investigated the self-assembly of(c-PNIPAM)-b-PCL in aqueous solution and thermal phase transition of c-PNIPAM corona within the micellar nanopartic

19、les,and compared to those of the linear diblock copolymer,(l-PNIPAM)-b-PCL,with comparable molecular weight and composition.The temperature-dependent release profiles from drug-loaded micelles of(c-PNIPAM)-b-PCL and(l-PNIPAM)-b-PCL were also explored in detail.2.We report on the fabrication of therm

20、oresponsive biohybrid double hydrophilic block copolymer(DHBC)via cofactor reconstitution approach.PNIPAM bearing ABSTRACT Va porphyrin moiety at the chain terminal,PPIXZn-PNIPAM,was synthesized via the combination of atom transfer radical polymerization(ATRP)and click chemistry.The subsequent cofac

21、tor reconstitution process between apomyoglobin and PPIXZn-PNIPAM afforded well-defined myoglobin-b-PNIPAM protein-polymer bioconjugates.Behaving as typical responsive DHBCs,the obtained myoglobin-b-PNIPAM biohybrid diblock copolymer exhibits thermo-induced aggregation behavior in aqueous solution d

22、ue to the presence of thermoresponsive PNIPAM block.Moreover,we also reported the facilely fabrication of well-defined protein-polymer bioconjugates with different chain architecture,and investigated their binding steric crowding effects.First,a series of biotinylated homopolymers and diblock copoly

23、mers with varying architecture and molecular weight were synthesized via a combination of ATRP and click chemistry.The locations of biotin in polymer chains were precisely varied:at the chain end or in the middle of homopolymer,at the chain end or the junction point of diblock copolymer.Taking advan

24、tage of the special interaction between avidin and biotin,we facilely fabricated star polymers,star block copolymers,and heteroarm star polymers.However,as the hydrophilic biotinylated polymers dissolved molecularly in aqueous media and existed as extended random coil,the binding course between biot

25、in and avidin would experience steric crowding effect in a certain extent.The effects of the DP of biotinylated polymer,and the location of biotin bound to the polymer chain on the conjugation efficiency were investigated in detail via standard avidin/HABA assays.3.We report on the fabrication of fl

26、uorescent pH-sensing organic/inorganic hybrid mesoporous silica nanoparticles(MSN)capable of tunable redox-responsive release of embedded guest molecules.Random copolymers composed of N-acryloxysuccinimide(NAS),oligo(ethylene glycol)monomethyl ether methacrylate(OEGMA),and 1,8-naphthalimide-based fl

27、uorescent pH-sensing monomer(NaphMA)were anchored at the surface of MSN via surface-initiated reversible addition-fragmentation chain transfer(RAFT)polymerization.The obtained hybrid MSN exhibits excellent water dispersibility and can act as sensitive fluorescent pH probe in the range of pH 4-8 due

28、to the presence of NaphMA moieties.After loading with model drug molecules,rhodamine B(RhB),and crosslinking the polymer brushes with cystamine,the redox-responsive release of encapsulated guest molecules from organic/inorganic MSN can be ABSTRACT VIfacilely tuned by varying the concentrations of ex

29、ternally added dithiothreitol(DTT).In another case,we report on the fabrication of pH-disintegrable polyelectrolyte multilayer-coated MSN capable of triggered co-release of cisplatin and model drug molecules.The outer polyelectrolyte multilayer was assembled from permanently cationic polyelectrolyte

30、,poly(allyl amine hydrochloride)(PAH),and negatively charged polyelectrolyte composed of N,N-dimethylacrylamide(DMA)and 3,4,5,6-tetrahydrophthalic anhydride-functionalized N-(3-aminopropyl)methacrylamide(TPAMA),which exhibits pH-induced charge conversion characteristics.Model drug molecule RhB was l

31、oaded into the interior mesopores of amine-functionalized MSN at first,this was followed by the layer-by-layer(LBL)deposition of P(DMA-co-TPAMA)and PAH at the outer surface of MSNs to effectively block the mesopore entrances.For cisplatin loading,it was mixed with the aqueous solution of P(DMA-co-TP

32、AMA)and embedded into the polyelectrolyte multilayer in the LBL assembly process.The structural stability of TPAMA moieties within the negatively charged pH-triggerable charge conversion polymer,P(DMA-co-TPAMA)is highly pH-dependent,i.e,stable under neutral media and hydrolyzed into positively charg

33、ed N-(3-aminopropyl)methacrylamide(APMA)moieties in weakly acidic media.Thus,the subtle alteration of solution pH from 7.4 to 5-6 can lead to the disintegration outer polyelectrolyte multilayers,accompanied with the co-release of cisplatin and RhB.4.We report on the fabrication of core cross-linked(

34、CCL)micelles possessing thermoresponsive cores and their application as sensitive and selective ratiometric Hg2+probes with thermo-tunable detection efficiency.Well-defined DHBC bearing naphthalimide-based Hg2+-reactive moieties(NUMA,4),PEO-b-P(NIPAM-co-NAS-co-NUMA),was synthesized via RAFT polymeri

35、zation,where PEO represents poly(ethylene oxide).The obtained DHBC can self-assemble into core-shell nanoparticles possessing thermoresponsive PNIPAM cores.After core cross-linking of the micellar nanoparticles formed at elevated temperatures,structurally stable CCL micelles with well-solvated PEO c

36、oronas and thermoresponsive cores embedded with Hg2+-reactive NUMA moieties were obtained.Upon Hg2+addition,the aqueous dispersion of CCL micelles exhibit a colorimetric transition from yellowish to colorless and a fluorometric emission transition from green to bright blue.The Hg2+-sensing capabilit

37、y of ABSTRACT VIIPEO-b-P(NIPAM-co-NAS-co-NUMA)unimers and CCL micelles at temperatures below and above the critical phase transition temperature was then determined and compared in detail.The fluorescence imaging assay of Hg2+ions in living cells was also investigated.In the last section,we reported

38、 on the synthesis of well-defined thermoresponsive polymers respectively labeled with fluorescence resonance energy transfer(FRET)pairs at chain middle and terminals,which can act as dual ratiometric fluorescent probes for pH and temperatures.Starting from difunctional initiator containing 7-nitro-2

39、,1,3-benzoxadiazole(NBD)moiety,the ATRP process of OEGMA and di(ethylene glycol)monomethyl ether methacrylate(DEGMA),and the subsequent terminal group functionalization with RhB-ethylenediamine derivative afforded NBD-P(OEGMA-co-DEGMA)-RhB2,which were labeled with FRET donor(NBD)and acceptor moietie

40、s(RhB)at the chain middle and terminals of the thermoresponsive polymer.The fluorescence emission of terminal RhB functionalities is highly pH-dependent,i.e,non-fluorescent in neutral or alkaline media(spirolactam form)and highly fluorescent in acidic media(ring-opened acyclic form),thus the off/on

41、switching of FRET process can be facilely modulated by solution pH.Moreover,at acidic pH and highly dilute conditions,the thermo-induced chain collapse and extension of NBD-P(OEGMA-co-DEGMA)-RhB2 can effectively modulate the spatial distance between FRET donor and acceptor moieties,leading to promin

42、ent changes in fluorescence intensity ratios.The incorporation of one FRET donor and two pH-switchable acceptors at the chain middle and terminals of thermoresponsive polymers allows for the effective off/on switching and the modulation of efficiency of FRET processes by dually playing with solution

43、 pH and temperatures.Key Words:Controlled/Living Radical Polymerization,Click Chemistry,Stimuli-Responsive,Double Hydrophilic Block Copolymer(DHBC),Self-Assembly,Bioconjugate,Mesoporous Silica Nanoparticles(MSN),Controlled Drug Release,Sensor,Ratiometric Fluorescent Probes 目 录 VIII目 录 目 录 摘摘 要要 I AB

44、STRACT IV 目目 录录 VIII 第一章第一章 绪论绪论1 1.1 结构规整聚合物的可控合成 1 1.1.1 原子转移自由基聚合 1 1.1.2 可逆加成-断裂链转移聚合 10 1.1.3 点击化学 5 1.2 响应性嵌段共聚物的超分子自组装 7 1.2.1 两亲性嵌段共聚物 7 1.2.2 双亲水性嵌段共聚物 10 1.2.2.1 温度敏感的水溶性聚合物10 1.2.2.2 pH 敏感的水溶性聚合物 12 1.2.3 生物杂化嵌段共聚物 13 1.3 基于响应性聚合物纳米载体的化学传感器 14 1.4 无机/聚合物杂化纳米载体的药物控制释放15 1.5 本论文的研究工作 17 参考文

45、献18 第二章第二章 两亲性蝌蚪形环两亲性蝌蚪形环-线聚合物的合成及其药物释放性能研究线聚合物的合成及其药物释放性能研究 33 2.1 概述 33 2.2 实验部分 39 2.2.1 原料及试剂 39 2.2.2 样品合成 40 2.2.3 仪器与表征 44 2.3 结果与讨论 44 目 录 IX2.3.1 蝌蚪形环-线嵌段共聚物(c-PNIPAM45)-b-PCL60的合成 44 2.3.2 蝌蚪形环-线嵌段共聚物(c-PNIPAM45)-b-PCL60的组装行为研究 49 2.3.3 蝌蚪形环-线嵌段共聚物(c-PNIPAM45)-b-PCL60的药物负载与释放 51 2.3.4 蝌蚪形环

46、-线嵌段共聚物(c-PNIPAM45)-b-PCL60的生物毒性测试 52 2.4 小结 53 参考文献54 第三章第三章 聚合物聚合物-蛋白质缀合物的合成及其组装性质研究蛋白质缀合物的合成及其组装性质研究 61 3.1 引言 61 3.2 辅助因子重构构筑双亲水性热敏聚合物-蛋白质嵌段共聚物61 3.2.1 概述 61 3.2.2 实验部分 66 3.2.2.1 原料及试剂 66 3.2.2.2 样品合成 110 3.2.2.3 仪器与表征 69 3.2.3 结果与讨论 70 3.2.3.1 双亲水性热敏感嵌段共聚物 myoglobin-b-PNIPAM 的合成70 3.2.3.2 双亲水性

47、热敏感嵌段共聚物 myoglobin-b-PNIPAM 的组装76 3.3 不同链构造聚合物-蛋白质缀合物的构筑及其立体位阻效应78 3.3.1 概述 78 3.3.2 实验部分 82 3.3.2.1 原料及试剂 82 3.3.2.2 样品合成 83 3.2.2.3 仪器与表征 86 3.3.3 结果与讨论 87 3.3.3.1 生物素功能化不同链构造聚合物的合成87 3.3.3.2 生物素功能化不同链构造聚合物与 avidin 之间结合系数的测定95 3.4 小结 97 参考文献97 第四章第四章 有机有机/无机杂化纳米粒子的可控药物释放无机杂化纳米粒子的可控药物释放 105 4.1 引言

48、105 4.2 基于杂化多孔硅的 pH 荧光探针及其氧化还原可控的药物释放 105 目 录 X4.2.1 概述 105 4.2.2 实验部分 110 4.2.2.1 原料及试剂 110 4.2.2.2 样品合成 110 4.2.2.3 仪器与表征 113 4.2.3 结果与讨论 113 4.2.3.1 pH 响应性无机/有机杂化多孔硅的制备 113 4.2.3.2 多孔硅表面交联及可控释放 119 4.2.3.3 杂化多孔硅的 pH 荧光检测性质121 4.3 pH 可电荷反转电解质多层膜修饰多孔硅的合成及其药物释放 124 4.3.1 概述 124 4.3.2 实验部分 128 4.3.2.

49、1 原料及试剂 128 4.3.2.2 样品合成 129 4.3.2.3 仪器与表征 130 4.3.3 结果与讨论 131 4.3.3.1 聚电解质多层膜修饰的无机/有机杂化多孔硅的制备131 4.3.3.2 杂化多孔硅的 RhB 和抗癌药物顺铂的负载及其 pH 诱导可控释放 136 4.4 小结 138 参考文献139 第五章第五章 环境响应性聚合物基化学传感器的合成及其性质研究环境响应性聚合物基化学传感器的合成及其性质研究 147 5.1 引言 147 5.2 基于热敏核交联胶束的比率型荧光 Hg2+探针147 5.2.1 概述 147 5.2.2 实验部分 154 5.2.2.1 原料

50、及试剂 154 5.2.2.2 样品合成 154 5.2.2.3 仪器与表征 156 5.2.3 结果与讨论 157 5.2.3.1 Hg2+响应 PEO-b-P(NIPAM-co-NAS-co-NUMA)核交联胶束的制备 157 5.2.3.2 基于核交联胶束 Hg2+离子传感器的检测性能表征 162 目 录 XI5.2.3.3 核交联胶束 Hg2+离子传感器的选择性 170 5.3 基于热敏聚合物的 pH 和温度的双重比率型探针 172 5.3.1 概述 172 5.3.2 实验部分 178 5.3.2.1 原料及试剂 178 5.3.2.2 样品合成 178 5.3.2.3 仪器与表征