【作者】 孙毅毅；

【导师】 侯世祥；

【作者基本信息】 四川大学 ， 药剂学， 2005， 博士

【摘要】 近年来,具有“隐形”功能的非肝位靶向长循环纳米粒的研究,已逐渐成为目前国内外纳米给药系统研究中活跃的热点领域,其中,共价结合亲水性高分子材料的隐形纳米粒载体材料的合成和应用研究越来越广泛。20世纪90年代,根据两亲聚合物分子自聚集现象和机理设想出的自聚集超分子复合物,作为一种具有长柔性链的亲水表面的新颖的隐形NP给药系统载体被广泛研究。这些两亲聚合物分子同时含有亲水(多数为PEG、PEO)和疏水基团,有类似表面活性剂的、在溶液中自动聚合形成亲水外壳、疏水内核组成的稳定的空间核-壳胶束结构。这种用聚合物自聚集而成的胶束纳米粒作为药物载体具有长柔性链的亲水表面,能够防止血浆中调理素的吸附,从而躲避网状内皮系统的识别与吞噬,易于实现长循环;同时由于释放共聚链段的速率较慢,故在体内的滞留时间长,毒副作用大大降低,生物相容性较小分子表面活性剂好,且能增强药物对生理屏障及肿瘤组织血管壁的渗透(EPR效应),促进细胞内药效发挥,在控制药物释放、达到靶向释放等方面展现出优良的性能,具有远大的发展前景。有良好的生物相容性和一定肿瘤亲和性的天然碱性多糖壳聚糖,本身带正电荷,将其制备成CS-PEG共聚物,进而制备成CS-PEG聚合物自聚集胶束纳米粒,则具有了不被体内调理素吸附的亲水表面、长柔性链及正电荷的隐形纳米粒的所有优势,是隐形纳米粒的理想载体材料。更多还原

【Abstract】 The "stealthy" non-liver targeting long circulating nanoparticles has became one of the research focuses in drug delivery systems. The synthesis and application of covalently linked hydrophilic polymers as the carrier for stealthy nanoparticles has drawn much attention. In the 90’s of the 20^th Century, the self-assembling supramolecular complexes were widely researched as a novel carrier for stealthy nanoparticles. The carrier was devised according to the self-assembling phemonenon of amphoteric polymers and the hydrophilic surface of the novel carrier has long limp chains. Those amphoteric polymers assembly automatically in water and form a dimensionally stable structure with hydrophilic crust and hydrophobic core. Those micelle nanoparticles formed by the self- assembly of polymers have hydrophilic surfaces with long limp chains. The structure of the micelle nanoparticles make them difficult to be absorbed by the opsonin in plasma, and helps them to evade the identification and absorption of reticuloendothelial system. So it is easier for this micelle nanoparticles to achieve long cirlulation. The micelle nanoparticles has low toxicity, good bio-compatibility and promoted permeability to physiological barriers and the vessel walls of tumor tissues. Chitosan is a positive charged natural alkaline polysaccharide with good bio-compatibility and moderate tumor affinity. The self-assembling polymer micelle nanoparticles made of the copolymer ofchitosan and PEG has positive charged hydrophilic surface and long limp chains, and it is an ideal carrier for stealthy nanoparticles.The graft copolymer of chitosan（CS） and MPEG was synthesized as the carrier of low toxic non-liver targeting stealthy micelle nanoparticles （MNP）, and it’s stability was studied. Norcantharidin（NCTD） was selected as the model drug and the micelle nanoparticles were made by norcantharidin and the copolymer ofchitosan and MPEG （NCTD-CS-MPEG- MNP）. The nanoparticles made by norcantharidin and chitosan （NCTD-CS-NP） were used as the control group. The pharmaceutical characteristics, stability, in vitro drug release, in vivo distribution and in vivo pharmacokinetics of the NCTD-CS-MPEG- MNP and NCTD-CS-NP were studied, and the long circulating characteristics of NCTD-CS-MPEG- MNP was researched.This research works is composed with 4 parts: the synthesis of the graft copolymer of chitosan-MPEG and its identification and stability; the preparation of NCTD-CS-NP and NCTD-CS-MPEG-MNP and their pharmaceutical characteristics; the in vitro drug release and stability of NCTD-CS-NP and NCTD-CS-MPEG-MNP; in vivo distribution and in vivo pharmacokinetics of the NCTD-CS-MPEG- MNP and NCTD-CS-NP in mice.With CS（MW.200000, de-acetyl rate>99%） and MPEG5000 as the materials, MPEG was activated by 1, l’carbonylediimidazole. This reaction was gentle and usually was used in protein-PEG. The activated MPEG was made with activation rate of 109% by choosing the best solvent, ratio of materials and reaction time, and the solubility or activation rate and recovery rate were used as the guidelines. Then, the activated MPEG reacted with primary amino groups ofchitosan and the graft copolymer of chitosan-MPEG was obtained through two steps by choosing the best solvent, reaction temperature, reaction time and ratio of materials and with the graft rate, determined by gravimetric method and indirect method, as the guidelines. The structure of the copolymer was identified with FT-IR, ’H-NMR and graft rate of the copolymer was determine to be 11% by gravimetric method, indirect method and ’ H-NMR. The graft rate agreedwith the PEG content of classical stealth nanoparticles materials. X-ray diffraction and DSC proved that the crystallinity of the copolymer raised and the copolymer had a tendency to form self-assembling micelle. The copolymer was stable at room temperature and was a promising carrier for stealthy nicelle nanoparticles. It was not reported at home and overseas that the graft copolymer of CS-MPEG, with the PEG content of classical stealth nanoparticles materials, was synthesized through two steps.It was not reported that NCTD-CS-NP and NCTD-CS-MPEG-MNP were prepared by chelation with metallic ions. The optimum pH, type and quantity of disperant. FeJ" and cosolventand, quantity of solidifying maerial. reaction temperature, reaction time and added drug were chosen by single factor methos. The obtained NCTD-CS-NP and NCTD-CS-MPEG-MNP were regular sphere with same particle size. The drug-loading rate, entrapment rate, average particle size and zeta potential of NCTD-CS-NP were 2.47%, 78.40%, 133nm and 15.92mV respectively. The drug-loading rate, entrapment rate, average particle size and zeta potential of NCTD-CS-MPEG-MNP were 1.21%, 72.33%, 129nm and 18.29mV respectively. The NCTD-CS-NP and NCTD-CS-MPEG-MNP were stable when they were stored for 3 months. Both had the similar drug-loading rate, entrapment rate, shape, particle size and zeta potential compared with the 0 month.In vitro experiments proved that the drug release of NCTD injections was not influenced by pH, the NCTD was released completely within lh. The drug release from NCTD injections accorded with the first class kinetic equation. The drug release from NCTD-CS-NP and NCTD-CS-MPEG- MNP was sustained evidently and accorded with the model of bi-exponent and bi-phase kinetics. The drug release from NCTD-CS-NP and NCTD-CS-MPEG- MNP decreased with the increase of pH.The results of in vivo study with mice showed that compared with NCTD injections, the distribution of NCTD-CS-NP and NCTD-CS-MPEG- MNP in mice was different. The difference are showed below:1) The plasma half-life and total targeting coefficient of NCTD-CS-MPEG-MNP was 67 folders and 23.5 folders of that of NCTD. The long-circulation characteristics was evident. 2) The total targeting coefficient and relative distribution efficiency showed that the liver affinity of NCTD-CS-NP was increased and NCTD-CS-MPEG- MNP was decreased. 3) Compared with NCTD. the targeting ability of NCTD-CS-NP to marrow increased, but its targeting ability to brain, tumor and plasma decreased. Compared with NCTD, the targeting ability of NCTD-CS-MPEG- MNP to brain, tumor and marrow increased and the total targeting coefficient of NCTD-CS-MPEG- MNP was 7.4, 8.6 and 9.1 folders of that of NCTD. 4) NCTD-CS-NP and NCTD-CS-MPEG-MNP decreased the distribution of NCTD in heart, lung and kidney, lowered the toxic effect of NCTD to these viscera. The results further showed that NCTD-CS-NP and NCTD-CS-MPEG- MNP can improve the efficacy and decrease the toxicity. The obvious long circulation and targeting to marrow of NCTD-CS-MPEG-MNP proved that it was possible to improve the efficacy of NCTD in treating leukaemia.The graft copolymer of chitosan and MPEG could self-assemblied into micelle nanoparticles with hydrophilic surface and hydrophobic core have evident "stealthy" effect. The carrier has greaer molecule weight and particle size, thus made it difficult to be excreted by kidney and prolonged the circulating time of drugs in blood. The long circulating micelle nanoparticles may promote the effect of NCTD in leukemia treatment. Our research works provided certain theoretical base for study of long circulating micelle nanoparticles made of macromolecular graft copolymers, and made it possible to utilize macromolecular self-assembling micelle nanoparticles as a drug delivery system.更多还原