Investigation Of Micellization And Vesiculation Of Conjugated Linoleic Acid By Self-assembly And Self-crosslinking Strategy

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.