Abstract
In the system of polymeric micelle, it has been widely applied to the drug delivery system (DDS). This research aims to develop and construct a multifunctionally mixed micelle which comes from mixing two different diblock copolymers, and can be used as drug carriers. The main points of this research are developing new system, materials and programs to construct polymer with polymer-mixed and nano-sized micelle. First, combine methoxy poly(ethylene glycol)-block-poly(D,L-lactide)(mPEG-b-PLA, DiblockⅠ) with methoxy poly(ethylene glycol)-block-poly(N-(2-hydroxypropyl) methacrylamide-co-lactate)(mPEG-b-P(HPMA-co-Lac), DiblockⅡ) or methoxy poly (ethylene glycol)-block-poly (N-(2-hydroxypropyl) methacrylamide-co-lactate- N-(2-hydroxypropyl) methacrylamide-co-L-Histidine)(mPEG-b-P(HPMA-co-Lac-HPMA-co-His), DiblockⅢ) to prepare the mixed micelle. DiblockⅠhas critical micelle concentration (CMC) property. DiblockⅡ and DiblockⅢ have critical micelle temperature (CMT), critical micelle concentration (CMC), and thermal sensitive properties. In addition, DiblockⅢ also has pH sensitivity property due to L-Histidine. DiblockⅠ respectively mixed with DiblockⅡ and DiblockⅢ to prepare core-shell structure and nano-size mixed micelle via hotshock protocol. Such micelle has well biocompatibility, biodegradability, uniform size about 50-100nm, and narrow polydispersity (PDI) below 0.2 at 37℃ buffer solution. DiblockⅠ stabilize and compact the structure against collapsing in blood because of micelle concentration shrinking. The temperature- and pH-sensitivity properties help micelle collapsing and dug releasing by the different temperature and pH in different tissues. The side chain groups of lactate can hydrolysis at physiological condition, contributing to the core of micelle hydrophilization, and upper the lower critical solution temperature (LCST). The controlled instability of micelle improves slowly in vivo degradation. Owing to these advantages, the mixed micelle has a candidate as drug carriers for cancer therapy. The traits of multifunctional mixed micelle are as follow. First, HPMA-co-Lac is the hydrophobic segment with temperature-sensitivity, providing a reservoir for hydrophobic drugs. When the temperature of the surroundings changed, the lower critical solution temperature move to high temperature due to the hydrolysis of lactate boosting, the behavior impel to micelle collapsing and drug releasing. Secondly, HPMA-co-Boc-His is pH-sensitive copolymer. When pH of the surroundings below pKa of Histidine, Histidine was protonated and positive charge, leading to the electronic repulsion and hydrophobic interaction of micelle decreased, and dug released. Finally, when the concentrations of copolymers are higher than CMC, copolymers draw others and aggregated, then form nano-micelle. By the above of advantages, we can double control nano-micelle to release drug. When the temperature and pH of the surroundings changed, the side chain group hydrolysis and contribute to the core of micelle changed from hydrophobic to hydrophilic, after all, the structure of the micelle collapsed and released drug. The temperature- and pH- sensitivity properties of the mixed micelle can be measured by UV-Vis spectrometer at 542nm and Zetasizer. The lower critical solution temperature occurred at 3-30℃ and gradually increased reducing environment pH. Furthermore, the use of pyrene as probe for fluorescence spectroscopic measurement could observe that the I337/I335 decreased with raising surrounding temperature, indicating that probe diffused from aqueous phase to the core of mixed micelle. The characteristics and morphologies of mixed micelle were analyzed from 1H-NMR, DLS, zeta potential, fluorescence spectrometer, UV-Vis spectrometer, GPC, and TEM. Additionally, Doxorubicin (Dox) was incorporate into mixed micelle for cancer therapy. In neutral surroundings, the release of Dox from mixed micelle was less. On the contrast, a significant release of Dox was observed in acidic surroundings about pH<6. The amount of drug released from mixed micelle was isolated from mixed micelle buffer solution by ultra-filtration and measured by UV-Vis spectrometer at 485nm in a time-course produre. The result of confocal laser scanning microscopy (CLSM) observation indicated that the loading drug successfully released in the acidic organelles due to the deformation of the micelle structure. Above all, the release of drug from mixed micelle could be accurately controlled by pH changes. From these results, the innovative mixed micelle showed high potential for drug carrier in intracellular drug delivery. Keyword: Metastatic cancer therapy, block copolymer, mixed micelle, hot shock protocol, dual controlled release, pH sensitivity, temperature sensitivity, CMT, CMC, LCST,