【作者】 周华锋；

【导师】 夏强；

【作者基本信息】 东南大学 ， 生物医学工程， 2015， 博士

【摘要】 心血管疾病(Cardiovascular disease, CVD)已成为全球范围导致人类死亡最主要原因之一,因此心血管靶向给药技术的研究对于心血管疾病的治疗具有极其重要的意义。本论文以辅酶Q10(Coenzyme Q10, CoQ10)为药物模型,设计并制备聚乙二醇1000维生素E琥珀酸酯(d-alpha-tocopheryl polyethylene glycol succinate, TPGS)及其阳离子氨基酸化衍生物修饰的系列CoQ10纳米乳(CoQ10-loaded Nanoemulsions, CoQ10-NEs、 CoQ10-NEs、CoQ10-NEs-TPGS-His、CoQ10-NEs-TPGS-Lys),并对上述四种辅酶Q10纳米乳制剂进行了细胞水平和在体水平的心肌靶向作用研究,为心肌靶向CoQ10-NEs的应用开发奠定基础。在纳米药物制备方面,本论文通过热熔乳化高压均质法制备由CoQ10、中碳链甘油酯(Caprylic/capric triglyceride, GTCC)及卵磷脂组成的CoQ10-NEs,并对其进行粒径、表面电位、包封率、稳定性及相变行为等表征,重点考察脂质比(CoQ10/GTCC)与粒径对CoQ10的结晶度的影响。结果表明,本方法制备的CoQ10-NEs形貌均为规整的球形,包封率均大于98%,CoQ10-NEs中CoQ10的光解速率较溶液中CoQ10降低了80.2%,实验发现粒径是影响CoQ10结晶度的关键因素,通过粒径控制达到无GTCC的CoQ10-NEs制备的目的。在靶向修饰方面,本论文选择新型辅料TPGS并设计合成了组氨酸(Histidine, His)及赖氨酸(Lysine, Lys)阳离子化TPGS衍生物TPGS-His及TPGS-Lys,对通过熔融乳化高压均质法制备粒径～50 nm由CoQ10与卵磷脂组成的CoQ10-NEs分别表面修饰TPGS、TPGS-His及TPGS-Lys o经MALDI-TOF.’HNMR及FT-IR波谱数据验证上述衍生物合成正确,制剂学表征实验表明表面修饰后的纳米乳制剂其粒径、相变行为等制剂学特征均未发生显著变化。对于上述系列辅酶Q10纳米乳制剂,本论文以大鼠为动物模型进行了静脉注射后药代动力学和组织分布研究,重点考察TPGS、TPGS-His及TPGS-Lys对CoQ10-NEs体内药代动力学及组织分布行为的影响,评估其对CoQ10-NEs的心肌靶向的影响。结果表明,CoQ10-NEs-TPGS与CoQ10-NEs-TPGS-His具有心肌靶向作用,而CoQ10-NEs与CoQ10-NEs-TPGS-Lys无心肌靶向作用,说明TPGS及TPGS-His具有促进CoQ10-NEs的心肌靶向作用；同时考察粒径对CoQ10-NEs的心肌组织CoQ10含量的影响,结果表明通过减少粒径可以促进CoQ10-NEs的心肌靶向作用。此外,本论文亦以心肌细胞为细胞模型研究了上述系列辅酶Q10纳米乳制剂与心肌细胞的相互作用。即以香豆素-6(CM6)为荧光标记分别制备粒径与上述纳米乳制剂一致的载有CM6的CM6-CoQ10-NEs、CM6-CoQ10-NEs-TPGS、CM6-CoQ10-NEs-TPGS-His及CM6-CoQ10-NEs-TPGS-Lys,通过细胞摄取动力学研究不同表面修饰物对心肌细胞制剂摄取的影响。结果表明,阳离子TPGS-His及TPGS-Lys对CoQ10-NEs的细胞摄取具有显著的促进作用,可在半小时内迅速提高细胞内CM6的浓度,且TPGS-Lys促进作用大于TPGS-His,同时缺氧培养条件显著增强了心肌细胞对阳离子CM6-CoQ10-NEs-TPGS-His及CM6-CoQ10-NEs-TPGS-Lys的摄取；而非离子TPGS对CoQ10-NEs的细胞摄取具有抑制作用。通过内吞抑制干预实验研究表明,CoQ10-NEs和CoQ10-NEs-TPGS输运CM6部分通过clathrin内吞途径进入细胞,而CoQ10-NEs-TPGS-His与CoQ10-NEs-TPGS-Lys输运CM6通过非内吞途径进入细胞,提示上述细胞摄取实验的差异或由转运途径差异所致。进一步的激光共聚焦显微镜观察表明,四种CoQ10-NEs均可输运CM6广泛分布于细胞核外胞浆内,同时CoQ10-NEs促进了CM6在细胞内线粒体的靶向分布,且TPGS-His增强了CoQ10-NEs输运CM6进入线粒体内靶向分布能力,而TPGS及TPGS-Lys抑制了CoQ10-NEs输运CM6进入线粒体内分布。上述细胞水平和在体水平研究共同表明,设计合成的表面修饰物TPGS-His对于药物心脏靶向作用有显著促进作用,可作为有效的药物辅料应用与心肌靶向药物设计,同时由此设计合成的辅酶Q10纳米乳制剂(CoQ10-NEs-TPGS-His)针对心血管疾病的靶向给药具有潜在的临床应用价值。更多还原

【Abstract】 Cardiovascular disease (CVD) is one of the most common causes of death worldwide. Thus, the development of drug delivery systems targeting the cardiovascular system is of great importance for the treatment of CVD. In this study, we used coenzyme Q10 (CoQ10) as a model drug to design CoQ10-loaded nanoemulsions (CoQ10-NEs). In vitro and in vivo experiments were conducted to examine the effect of D-α-tocopheryl polyethylene glycol succinate (TPGS) and its cationic amino acid derivatives on CoQ10-NE myocardial targeting. These results will lay a foundation for the application and development of myocardial-targeted CoQ10-NEs.CoQ10-NEs composed of CoQ10, caprylic/capric triglyceride (GTCC), and lecithin were prepared by melt emulsification and high-pressure homogenization. The particle size, surface potential, encapsulation efficiency, stability, and phase transition behavior of the CoQ10-NEs were evaluated, with particular emphasis on the effects of CoQ10/GTCC and particle size on the crystallinity of CoQ10. Our results indicated that CoQ10-NEs prepared by this method have a regular, spherical morphology, with an encapsulation efficiency greater than 98%. The photolysis rate of CoQ10 in the CoQ10-NEs was reduced by 80.2%, as compared to CoQ10 in solution. Particle size was shown to be a key factor influencing CoQ10 crystallinity. Thus, preparation of CoQ10-NEs without GTCC can be implemented using particle size control.Next, cationic histidine (His) and lysine (Lys) derivatives of TPGS (TPGS-His and TPGS-Lys) were prepared and verified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF),1H nuclear magnetic resonance (NMR), and Fourier transfer infrared (FT-IR) spectral data. CoQ10-NEs (50 nm) composed of CoQ10 and lecithin were prepared by melt emulsification and high-pressure homogenization, followed by surface modification with TPGS, TPGS-His, and TPGS-Lys. In vivo experiments were performed in rats to assess the effect of TPGS, TPGS-His, and TPGS-Lys on CoQ10-NE pharmacokinetics, tissue distribution, and myocardial targeting. Our results showed that both CoQ10-NEs-TPGS and CoQ10-NEs-TPGS-His could target the myocardium, whereas CoQ10-NEs and CoQ10-NEs-TPGS-Lys did not. These data indicate that TPGS and TPGS-His can target CoQ10-NEs to the myocardium. Additionally, the effect of particle size on CoQ10 levels in the myocardial tissue was examined. Myocardial targeting of CoQ10-NEswas enhanced by reducing particle size.Finally, in vitro experiments were performed in a cardiomyocyte cell line. Fifty nanometer CM6-CoQ10-NEs, CM6-CoQ10-NEs-TPGS, CM6-CoQ10-NEs-TPGS-His, and CM6-CoQ10-NEs-TPGS-Lys were prepared with coumarin-6 (CM6) as a fluorescent tag, and their interactions with cardiomyocytes were evaluated. Kinetic analyses of cellular uptake showed that cationic TPGS-His and TPGS-Lys significantly promoted cellular uptake of CoQ10-NEs, rapidly increasing the concentration of intracellular CM6 within 30 min. Interestingly, TPGS-Lys had a stronger effect than TPGS-His. Additionally, hypoxic culture conditions significantly enhanced the uptake of cationic CM6-CoQ10-NEs-TPGS-His and CM6-CoQ10-NEs-TPGS-Lys by cardiomyocytes, whereas non-cationic TPGS had an inhibitory effect. Inhibition of endocytosis revealed that CoQ10-NEs and CoQ10-NEs-TPGS transported CM6 into the cell via the clathrin endocytic pathway, whereas CoQ10-NEs-TPGS-His and CoQ10-NEs-TPGS-Lys transported CM6 via a non-endocytic pathway. Laser confocal microscopy analyses revealed that the four CoQ10-NEs were widely distributed in the cell cytoplasm, outside the nucleus. Furthermore, CoQ10-NE uptake resulted in mitochondrial distribution of CM6 within the cell. Moreover, TPGS-His enhanced the ability of CoQ10-NEs to transport CM6 into the mitochondria, whereas TPGS and TPGS-Lys inhibited CM6 transport into the mitochondria.更多还原