纳米给药系统跨内耳圆窗膜转运机理的研究

发布时间：2018-03-21 16:06

本文选题：PLGA纳米粒　切入点：CS纳米粒　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：通过圆窗膜给药的内耳局部给药系统由于其有效性,非侵入性,绕过血迷路屏障以及降低在非靶向部位的毒性等优点越来越受到耳科学家的青睐。近年来,纳米粒给药系统在内耳局部给药方面的应用越来越广泛,其小粒径、高比表面积及高活性可以使纳米粒有效穿透体内的各种生物学屏障。目前有关于纳米药物在内耳的研究主要集中于内耳的组织分布,药动学以及药效学研究。但是圆窗膜作为纳米粒局部给药系统主要的生物学屏障,目前并没有对纳米粒与圆窗膜之间的相互作用进行研究。而了解清楚纳米粒跨越圆窗膜的转运机制的研究对于纳米粒的进一步合理化设计是非常关键的。聚乳酸羟基乙酸(PLGA)是FDA批准的可生物降解的药用辅料,在内耳纳米粒给药系统方面有着非常广泛的应用,采用乳化溶剂挥发法将香豆素-6包载入PLGA,制备香豆素-6标记的PLGA纳米粒,对其在圆窗膜的通透性及转运机制进行研究。豚鼠,鼓室注射PLGA纳米粒30 min后,分离出圆窗膜进行膜铺片操作,采用激光扫描共聚焦显微镜观察,发现香豆素-6的绿色荧光在圆窗膜中广泛分布,说明纳米粒可以进入圆窗膜;同时采集纳米粒给药后的外淋巴样本进行透射电镜观察发现纳米粒可以以完整形式进入外淋巴,说明了纳米粒鼓室给药后,可以穿越圆窗膜到达外淋巴空间。通过改变鼓室注射PLGA纳米粒的浓度,选用了0.01 g/ml,0.03 g/ml及0.09 g/ml三个浓度点对纳米粒穿越圆窗膜进行浓度依赖性考察,结果发现圆窗膜内的荧光量随着给药浓度的增加而增加。选用了10 min,30 min及60 min三个时间点,对PLGA纳米粒穿过圆窗膜进行时间依赖性考察,发现在给药30 min内,圆窗膜内的荧光量随着时间的增加而增加,但是在30 min后,随着时间的增加而降低。对鼓室注射PLGA纳米粒30 min的豚鼠圆窗膜进行透射电镜观察,与正常组的圆窗膜相比,给药组的圆窗膜中发现了大量的内吞及胞吐小泡,同时圆窗膜外上皮细胞之间的紧密连接并没有打开,因此可知PLGA跨越圆窗膜主要是通过内吞的方式进入细胞,在细胞内进行囊泡转运,而不是通过细胞旁路途经。采用抑制不同内吞途径的抑制剂,发现PLGA纳米粒主要是以小窝蛋白以及巨胞饮介导的内吞途径进入细胞。对细胞内的溶酶体进行荧光标记,采用激光扫描共聚焦共定位技术,对溶酶体及香豆素-6标记的PLGA纳米粒进行共定位,发现纳米粒摄取进入细胞后,会进入细胞的溶酶体。采用与高尔基体相关的抑制剂发现细胞内的内质网及高尔基体可以介导纳米粒的胞吐。对内耳疾病进行合理有效的治疗很大程度上取决于耳蜗内恒定的药物浓度。壳聚糖(Chitosan,CS)是一种生物可降解的聚合物,由于其粘附特性是一种优良的药物载体,可以增加纳米粒与圆窗膜的接触时间,同时壳聚糖具有打开上皮细胞间紧密连接的作用,可以增加纳米粒的细胞旁路途经。采用离子交联法制备载香豆素-6的CS纳米粒,鼓室给药后进行圆窗膜铺片,采用激光扫描共聚焦显微镜成像后得到的圆窗膜的3D重建图像以及外淋巴的透射电镜充分证明了壳聚糖纳米粒可以穿过圆窗膜进入外淋巴。对壳聚糖跨越圆窗膜进行时间依赖性考察,选取了5个给药时间点,10 min、30 min、1 h、2 h及4 h,结果显示在给药60 min以前,圆窗膜中香豆素-6的荧光量随着时间的延长而增加,但是在60 min到240 min的时间段内逐渐减弱;通过改变壳聚糖纳米粒给药的浓度,选用了3个浓度点5 mg/ml,10 mg/ml及20 mg/ml,发现圆窗膜中壳聚糖纳米粒的荧光量随着给药纳米粒浓度的增加而增加,呈现浓度依赖性。纳米粒进入上皮屏障有细胞内和细胞间两种途径,采用透射电镜对壳聚糖给药后的圆窗膜进行成像,发现壳聚糖纳米粒可以以细胞旁路及跨细胞两种方式进入圆窗膜。采用不同途径的内吞抑制剂处理圆窗膜发现壳聚糖纳米粒以小窝蛋白,网格蛋白及巨胞饮三种途径进入圆窗膜细胞,说明了壳聚糖纳米粒与圆窗膜细胞之间的非特异性相互作用。采用荧光共定位技术观察到了壳聚糖纳米粒在溶酶体的分布,高尔基体相关的抑制剂说明壳聚糖纳米粒可以在内质网及高尔基体的介导下胞吐出细胞。综上所述,本课题第一次直观证明了PLGA纳米粒及CS纳米粒确实可以穿过圆窗膜到达外淋巴,并阐明了纳米粒在圆窗膜的内吞,细胞内转运及胞吐过程的详细的机制,为制剂的进一步优化提供指导。
[Abstract]:Through the round window administration of inner ear local drug delivery system because of its effective, non-invasive, bypassing the blood labyrinth barrier and reduce the toxicity to the site has more and more experts in non target ear of all ages. In recent years, nanoparticles drug delivery system in the inner ear are used for local delivery of the more widely, the small size, high surface area and high activity can make nanoparticles efficiently penetrate various biological barrier in vivo. There are about nano drug in the inner ear research focused on the inner ear tissue distribution and pharmacodynamics pharmacokinetics. But the round window membrane nanoparticles as local drug delivery system biology barrier, there is no research on the interaction between the nanoparticles and the round window. The research and understand the transport mechanism of nanoparticles across the round window membrane is the key to design more reasonable nanoparticles The poly lactic co glycolic acid (PLGA) is a medicinal materials FDA approved biodegradable, are widely used in the inner ear of nanoparticle delivery system, by emulsion solvent evaporation method of coumarin -6 package in the PLGA, the preparation of coumarin -6 labeled PLGA nanoparticles, the research on permeability and transport mechanism RWM. Guinea pigs, intratympanic injection of PLGA nanoparticles after 30 min, isolated by round window membrane preparation operation, using laser scanning confocal microscopy, -6 green fluorescent coumarin found widely distributed in the round window, that nanoparticles can enter the round window; at the same time collected after administration of nanoparticles in peripheral lymphoid samples for transmission electron microscopy showed that the nanoparticles can be in complete form into the extralymphatic, illustrates the nanoparticles after intratympanic administration, can pass through the round window membrane to reach the external lymph space. By changing the tympanic injection of PLGA sodium Grain concentration, with 0.01 g/ml, 0.03 g/ml and 0.09 g/ml three concentration of nanoparticles were concentration dependent through round window inspection, found that the quantity of fluorescence in round window membrane increased with increasing concentration. By 10 min, 30 min and 60 min three time points of PLGA nanoparticles through the round window time dependent study, found in the administration of 30 min, the quantity of fluorescence in round window membrane increased with the increase of time, but after 30 min, decreased as time increased. The guinea pig RWM intratympanic injection of PLGA nanoparticles of 30 min in transmission electron microscopy, compared with the round window normal group, drug group to the round window was found in a large number of endocytosis and intracellular vesicles and spit, between the outer round window membrane epithelial cells connected closely and not open, so the PLGA across the round window membrane is mainly through endocytosis into cells, For vesicle transport in the cells, rather than by using different inhibitors. Cell bypass via the endocytic pathway is inhibited, PLGA nanoparticles mainly enter cells by caveolin and endocytic pathways mediated macropinocytosis. On intracellular lysosomes were labeled, using laser scanning confocal colocalization of technology. Co localization of lysosomes and coumarin -6 labeled PLGA nanoparticles, found nanoparticles uptake into the cell, will enter the cell lysosomes. The inhibitor associated with the Golgi endoplasmic reticulum and Golgi cells can be mediated by nanoparticles exocytosis. The drug concentration of inner ear diseases were reasonable and effective treatment depends largely on in the cochlea constant. Chitosan (Chitosan, CS) is a kind of biodegradable polymer, because of its adhesion is an excellent drug carrier, can increase The contact time of nanoparticles and round window membrane, and chitosan has opened the role of tight junctions between epithelial cells, can increase cell bypass via using CS nanoparticles. Nanoparticles prepared by ionic crosslinking method containing coumarin -6, after intratympanic administration of RWM flatmount using laser scanning confocal microscope imaging obtained after the round window the 3D image reconstruction and perilymphatic TEM proved that chitosan nanoparticles can pass through the round window into the extralymphatic. The chitosan cross round window membrane subjected to time-dependent study, selected 5 time points for administration, 10 min, 30 min, 1 h, 2 h and 4 h. The results showed that in to the medicine before 60 min, the amount of fluorescence of coumarin -6 in round window membrane increased with time, but in 60 min to 240 min within the time period gradually weakened; by changing the chitosan nanoparticles for drug concentration, choose 3 concentration At 5 mg/ml, 10 mg/ml and 20 mg/ml, it was found that the fluorescence quantity in round window membrane of chitosan nanoparticles increased with the increase of drug concentration of nanoparticles, in a dose-dependent manner. The nanoparticles into epithelial barrier cells within and between cells in two ways, by transmission electron microscopy of chitosan to round window membrane after Administration of imaging. Found that chitosan nanoparticles can enter the cells to bypass and cross RWM cells in two ways. The different pathways of endocytosis inhibitors found that chitosan nanoparticles to RWM caveolin, clathrin and macropinocytosis three ways to enter the round window membrane cell, illustrates the interaction between chitosan nanoparticles and cells of non round window specific. Using fluorescent colocalizationanalysis observed chitosan nanoparticles in lysosomal inhibitor distribution, the Golgi associated that chitosan nanoparticles in the endoplasmic reticulum and high er The matrix mediated exocytosis from the cell. To sum up, this is the first time to directly prove that PLGA nanoparticles and CS nanoparticles can pass through the round window membrane to reach the lymph and clarify the nanoparticles in round window membrane endocytosis, intracellular transport and exocytosis of the detailed mechanism, to provide guidance for the further optimization of preparation.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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