【作者】 樊晔；

【导师】 方云；

【作者基本信息】 江南大学 ， 应用化学， 2014， 博士

【摘要】 共轭亚油酸(CLA)是天然存在的一类不饱和脂肪酸，具有抗癌、抗粥状动脉硬化、减脂等多种有益生理活性。目前国内外对于CLA的关注点主要集中在将其作为食品功能因子和营养保健因子等方面。CLA除了具有有益生理活性之外，其分子结构中的羧酸基团具有pH刺激响应性赋予CLA自组装活性，同时其分子结构中的共轭双键能够提供可聚合位点赋予CLA自交联活性。然而，目前从分子自组装角度研究CLA的胶体与界面化学性质以至开发与拓展其在非食用领域的应用价值仍然尚未涉猎。因此，研究CLA的有序自组装，有助于构建多种尺度和形貌的CLA有序自组装体；探讨CLA有序自组装体的自交联行为，有助于通过合适的交联反应增强CLA有序自组装体的胶体稳定性，对于拓宽其在药物载体、纳米材料模板合成乃至小分子有序自组装体直观表征领域的应用前景非常有意义。近年来，脂肪酸自组装行为的研究主要集中在饱和脂肪酸、单不饱和脂肪酸和非共轭多不饱和脂肪酸，对于共轭不饱和脂肪酸的自组装行为研究仍然空白。本文选择CLA为研究对象，利用自组装-自交联策略研究其有序自组装体。因此本论文关注的第一个问题是：CLA具有怎样的自组装规律和胶束-囊泡形貌转换规律？脂肪酸的pH响应性能够使其在不同的pH环境中自组装形成多种尺度和形貌的自组装体。其中脂肪酸胶束和脂肪酸囊泡是两类重要的脂肪酸自组装体。然而脂肪酸自组装体的pH依赖性、二价离子敏感性甚至浓度敏感性导致其存在胶体稳定性差的缺陷，不仅妨碍了对脂肪酸自组装体结构的明确表征，而且对于脂肪酸自组装体的应用研究也十分有限。因此本论文关注的第二个问题是：在CLA自组装成胶束和囊泡的基础上，利用其较高的自交联活性能否通过可控自交联过程获得清晰的球形胶束图像和构建非pH敏感型囊泡？脂肪酸囊泡与传统包埋缓释载体脂质体的结构类似，两者包封率基本相当；与此同时脂肪酸囊泡的原料脂肪酸价廉易得，安全性更高，解决了脂质体中天然磷脂成分复杂并且难以合成质量上乘的磷脂分子的缺点。因此脂肪酸囊泡在食品添加和药物载体领域具有良好的应用前景。但是脂肪酸自组装体胶体稳定性差的缺陷，限制了它们在食品添加和药物载体等应用领域的发展；同时脂肪酸胶束的应用仍有待开发。因此本论文关注的第三个问题是：自组装-自交联技术可能会为CLA自组装体拓展怎样的应用前景？围绕以上问题，本论文开展了以下几方面的主要工作：1、共轭亚油酸的合成与表征采用尿素包合法和碱异构化法从天然红花籽油合成CLA。优化包合提取条件和异构化反应条件尽可能提高中间体亚油酸(LA)和目标产物CLA的产率和纯度。结合FT-IR、UV、1H NMR确认目标产物的结构，通过GC分析目标产物的纯度。实验结果表明，采用尿素包合法，以混合脂肪酸:尿素:95%乙醇=1:2.5:8(w/w/v)分离富集，使LA的含量由混合脂肪酸原料中72.36wt%提高至97.60wt%。然后以KOH:LA:乙二醇=1:2:3(w/w/v)条件在170oC下进行碱异构化反应4h合成CLA，含量达96wt%。2、共轭亚油酸的pH响应自组装和形貌变化规律采用表面张力法系统研究不同pH、温度和离子强度下CLA的表面物理化学性质和自组装行为，确认CLA自组装的推动力。通过考察不同温度下CLA的自组装行为，计算其胶束化/囊泡化热力学参数。并且采用酸碱平衡滴定实验分析不同形貌CLA自组装体形成的pH范围。实验结果表明，CLA在pH13.0-10.0区间内自组装成胶束(CLA-micelle)，在pH9.0-8.0范围内自组装成囊泡(CLA-ufasome)。而且比较CLA和文献报道的其它脂肪酸形成囊泡的适宜pH范围发现，除脂肪酸链长之外，脂肪酸分子中双键数目越多以及共轭性越强，形成囊泡的pH值越低。3、共轭亚油酸自组装体的自交联以兼具有益生理活性、pH响应自组装活性和较高自交联活性的CLA为分子砌块，首先通过简单的pH响应自组装获得共轭亚油酸胶束(CLA-micelle)，然后采用微干扰的紫外辐照诱导方式对CLA-micelle实施胶束内化学绑定，制得了3-5nm的稳定自交联CLA胶束(CLA-FAM)。比较未交联的CLA-micelle的冷冻透射电子显微镜(cryo-TEM)图像和自交联CLA-FAM的透射电子显微镜(TEM)图像发现，自交联CLA-FAM基本维持了CLA-micelle的原始形貌，进一步佐证了CLA-micelle的球形小胶束结构。采用类似的自组装-自交联技术，通过紫外辐照对共轭亚油酸囊泡(CLA-ufasome)实施化学绑定制得粒径为10-20nm、双层膜厚度约为2.0nm的非pH敏感的自交联CLA囊泡(CLA-FAV)。利用cryo-TEM观察未交联CLA-ufasome的形貌为20nm左右的囊泡结构，这表明紫外辐照制备的自交联CLA-FAV基本保持了CLA-ufasome的原始脆弱形貌。而且自交联CLA-FAV双层膜中CLA分子呈肩并肩方式排列而非传统的尾对尾方式；综合实验结果和文献报道，认为脂肪酸囊泡双层膜厚度与其中的脂肪酸分子排列方式有关，而双层膜中脂肪酸分子采取何种排列方式则主要取决于脂肪酸分子中双键的位置、数目和共轭程度而并不是脂肪酸分子链长。4、自交联共轭亚油酸自组装体的应用采用高温热聚合引发手段，引发CLA胶束和本体相同时发生自交联反应，制备得到以20nm的CLA球体为联结单元的独特网状结构聚共轭亚油酸(PCLA)；在此基础上，利用本实验室前期研究的极性聚合物表面氯金酸原位还原沉积方法，获得以CLA胶束为核的网状纳米金结构，而且该PCLA基网状纳米金比普通胶态金纳米颗粒具有更好的SERS效应和催化效果。实验结果表明，网状PCLA基底的模板作用和原位还原作用相结合是制得PCLA基网状纳米金的充分必要条件。本文开发了一种仅从简单的天然共轭不饱和脂肪酸出发，无需外加交联剂、化学还原剂和锚定试剂，无需预制备纳米金粒子，无需复杂的制备流程，就能获得聚合物基网状纳米金的方法。类似的，利用高温热聚合引发CLA-ufasome中CLA的共轭双键自交联实施囊泡内化学绑定，最终获得粒径为20-50nm、双层膜厚度为2.7±0.5nm的非pH敏感的自交联CLA-FAV。以两种不同聚合方式制得的自交联CLA-FAV为载体，包覆药物五氟尿嘧啶(5-FU)均表现出缓释效果。表明非pH敏感型自交联CLA-FAV在药物载体领域具有应用前景。更多还原

【Abstract】 Conjugated linoleic acid (CLA) is a natural unsaturated fatty acid with excellentanticarcinogenic, antiatherogenic and anti-inflammatory functions. Over the past decades,CLA has been only concerned as food or medicine ingredients. But CLA also has twocharacteristics beside the bioactivity. One is the self-assembling activity which is attributed tothe pH response of the carboxyl group in the CLA molecule; the other is the self-crosslinkingactivity which is because that the double bonds in the CLA molecule can providepolymerizable sites. However, up to date, studies on the colloidal properties of CLA from theperspective of molecular assembly and further developing their application in non-food fieldsstill have not been dabbled. Thus, it is necessary to investigate the self-assembly of CLA andthe self-crosslinking behavior of the CLA self-assemblies. Since the self-assembly of CLA isbenefit to construct various CLA self-assemblies with different sizes and morphologies, andthe self-crosslinking of the CLA self-assemblies is favorable for improving their colloidalstability. Moreover, it plays an important role in expanding the application prospect in drugdelivery, templating synthesis of nanomaterials and even direct observation of molecularassemblies.Recently, investigations of the self-assembling behaviors of fatty acids have beenfocused on that of saturated fatty acids, monounsaturated fatty acids and non-conjugatedpolyunsaturated fatty acids. However, the self-assembling behaviors of conjugatedunsaturated fatty acids are still not studied. In this dissertation, CLA is employed as theresearch object, and the micellization and vesiculation of CLA are investigated withself-assembly and self-crosslinking strategy. Therefore, the first concern in this dissertation is,how about the regulations of self-assembly and transformation from micelles to vesicles ofCLA?Fatty acids can self-assemble into various assemblies with different sizes andmorphologies due to their pH response. Among the self-assemblies, fatty acid micelle (FAM)and fatty acid vesicle (FAV) are two important assemblies of fatty acids. However, the directobservation and the applications of fatty acid assemblies are still hindered by the colloidalstability such as pH-, temperature-, divalent cation-and even concentration-sensitivity.Therefore, the second concern in this dissertation is, can we obtain an unambiguous image ofspherical micelle and construct a pH-insensitive vesicle through self-crosslinking in virtue ofthe self-crosslinking activity of CLA?The structure of FAV is similar to that of liposome, which is a common embedded andsustainable releasing system, and the encapsulation efficiency of FAV is equal to that ofliposome. Moreover, the ready availability of unsaturated fatty acids overcomes the weaknessof liposome, because the natural phospholipids are chemically heterogeneous and puresynthetic phospholipids are not yet available in reasonable quantities. Therefore, FAV has apromising prospect of application in the fields of food additives, drug delivery and sustainedrelease systems. However, the applications of FAV in the fields of food additives, drugdelivery and sustained release systems are still hindered by the colloidal stability. Likewise, the applications of FAM are unexplored. Thus, the third concern in this dissertation is, whichapplication will be expanded through self-assembly and self-crosslinking strategy?Aiming at the aforementioned concerns, this dissertation mainly focuses on thefollowing:1、Preparation and characterization of conjugated linoleic acidCLA was synthesized from safflower oil through urea inclusion and alkali isomerization.Optimizing the conditions of urea inclusion and alkali isomerization was to obtain linoleicacid (LA) and CLA with high yield and purity. The obtained CLA was characterized withFT-IR、UV、1H NMR and GC, respectively. The experimental results indicated that the contentof LA was up to97.60wt%from72.36wt%by urea inclusion when fatty acids mixture: urea:ethanol=1:2.5:8(w/w/v). CLA of96wt%were synthesized at170oC for4h when KOH:LA: ethylene glycol=1:2:3(w/w/v).2、The pH responsive self-assembly and transformation of various assemblies of conjugatedlinoleic acidThe surface activities and self-assembling behaviors of the semi-synthetic CLA weresystematically investigated by tensiometry method at different pH values, temperatures andionic strength. In addition to the surface activity parameters, the thermodynamic parametersof micellization or vesiculation of CLA at different pH were calculated according to theself-assembling behaviors of CLA based on the pseudo-phase separation model. And then, thesuited pH ranges for self-assembling of CLA assemblies were analyzed by acid-base titration.The experimental results indicated that CLA could self-assemble into micelles and ufasomesin the ranges of pH13.0-10.0and pH9.0-8.0, respectively. It was found that pH, temperatureand ionic strength obviously influenced the surface activities of CLA, but pH is the prominentfactor affecting on the self-assembling of CLA. Moreover, the common driving forces of themicellization and vesiculation of the CLA solution are entropy increase. Furthermore, it wasfound that besides the hydrophobic chain lengths the numbers and the conjugation degrees ofthe double bonds are the factors influencing the suitable pH ranges, that is, the suited pHrange depends on the hydrophobicity of the fatty acid molecule, and more double bonds andhigher conjugation degrees in a fatty acid molecule result in lower pH range for vesicleformation.3、The self-crosslinking of conjugated linoleic acid assembliesCLA-micelles were self-assembled in response to pH variation using CLA with tripleactivities as a new molecular building block. The stable self-crosslinked CLA micelles(CLA-FAM) with diameter of3-5nm were obtained by chemical tethering the CLA-micellein virtue of intra-micellar crosslinking initiated by moderate UV irradiation. Based oncomparison of the cryo-TEM image of the non crosslinked CLA-micelles and the TEM imageof the self-crosslinked CLA-FAM, it was found that the self-crosslinked CLA-FAMmaintained the original and vulnerable morphology of CLA-micelle and provided theevidence for the small micellar structure of CLA-micelles. The pH-insensitive self-crosslinked CLA vesicles (CLA-FAV) with diameter of10-20nm and bilayer thickness of~2.0nm were prepared by intra-ufasomal crosslinking ofconjugated double bonds in CLA molecules through UV irradiation. In particular, the noncrosslinked CLA-ufasome was vesicle with diameter of~20nm imaged by cryo-TEM. Theexperimental results indicated that the self-crosslinked CLA-FAV obtained by UV irradiationmaintained the original and vulnerable morphology of CLA-ufasome. In addition, based onthe comparison of the bilayer thicknesses of different fatty acids, it was pointed out that themolecular arrangement in the bilayer membrane of the CLA-ufasome is side-by-side modelrather than the traditional tail-to-tail model. Moreover, it was found that the numbers andthe conjugation degrees of double bonds in an unsaturated fatty acid molecule rather than thehydrophobic chain length are the key factors to control the bilayer thickness of the ufasome.4、The application of the self-crosslinked CLA assembliesThe networked poly(conjugated linoleic acid)(PCLA) aggregates were prepared at highpH through simple molecular self-assembly and thermal polymerization using CLA asmonomer. The morphology of the PCLA aggregates was a unique network structure with CLAspherical micelle of~20nm as junction unit, imaged by TEM. Subsequently, the Aunano-network with core of CLA micelle was synthesized through in situ reduction of gold atthe surface of the PCLA network. Moreover, the Au nano-network based on PCLA possessedbetter SERS and catalytic effect than the colloidal Au nanoparticles. It was suggested that it isnecessary to combine PCLA network with in situ reduction of gold at the surface of the PCLAnetwork in the synthesis process of the Au nano-network based on PCLA. The experimentalresults indicated that it explores a simple route for synthesis of Au nano-network based onpolymer sbustract which is only from a natural conjugated unsaturated fatty acid withoutcross-linker, reducing agents, anchoring agents and Au pre-nanoparticles.Similarly, the other pH-insensitive self-crosslinked CLA-FAV were prepared byintra-ufasomal crosslinking of conjugated double bonds in CLA molecules through thermalpolymerization. The size distribution and the bilayer thickness of the self-crosslinkedCLA-FAV were imaged by TEM and being20-50nm and2.7±0.5nm, respectively.Furthermore, the experimental results of in vitro release of5-fluorouracil from the two aboveself-crosslinked CLA-FAV showed that the release process was slow and sustainable,indicating the pH-insensitive self-crosslinked CLA-FAV has a promising prospect ofapplication in the fields of food additives and drug delivery systems.更多还原