【作者】 万学娟；

【导师】 刘世勇；

【作者基本信息】 中国科学技术大学 ， 高分子化学与物理， 2011， 博士

【摘要】 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-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。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β-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β-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Hg²⁺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-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pH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和温度的双重比率型检测。更多还原

【Abstract】 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 catalysis, 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 novel 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, 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 solution. 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. Specifically, 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（ε-caprolactone） （PCL） and thermoresponsive macrocyclic PNIPAM via 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 nanoparticles, 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 thermoresponsive biohybrid double hydrophilic block copolymer （DHBC） via cofactor reconstitution approach. PNIPAM bearing a porphyrin moiety at the chain terminal, PPIXZn-PNIPAM, was synthesized via the combination of atom transfer radical polymerization （ATRP） and click chemistry. The subsequent cofactor 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 due 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 copolymers 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 advantage 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 biotin 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 fluorescent 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 fluorescent 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 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 facilely tuned by varying the concentrations of externally 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, 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 loaded 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-TPAMA） 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 charged 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 （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 polymerization, 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 coronas 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 capability of PEO-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 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-2,1,3-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 moieties （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 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 prominent 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 pH and temperatures.更多还原