本文選題：PLGA納米粒　切入點：CS納米粒　出處：《廣東藥科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：通過圓窗膜給藥的內(nèi)耳局部給藥系統(tǒng)由于其有效性,非侵入性,繞過血迷路屏障以及降低在非靶向部位的毒性等優(yōu)點越來越受到耳科學(xué)家的青睞。近年來,納米粒給藥系統(tǒng)在內(nèi)耳局部給藥方面的應(yīng)用越來越廣泛,其小粒徑、高比表面積及高活性可以使納米粒有效穿透體內(nèi)的各種生物學(xué)屏障。目前有關(guān)于納米藥物在內(nèi)耳的研究主要集中于內(nèi)耳的組織分布,藥動學(xué)以及藥效學(xué)研究。但是圓窗膜作為納米粒局部給藥系統(tǒng)主要的生物學(xué)屏障,目前并沒有對納米粒與圓窗膜之間的相互作用進行研究。而了解清楚納米粒跨越圓窗膜的轉(zhuǎn)運機制的研究對于納米粒的進一步合理化設(shè)計是非常關(guān)鍵的。聚乳酸羥基乙酸(PLGA)是FDA批準(zhǔn)的可生物降解的藥用輔料,在內(nèi)耳納米粒給藥系統(tǒng)方面有著非常廣泛的應(yīng)用,采用乳化溶劑揮發(fā)法將香豆素-6包載入PLGA,制備香豆素-6標(biāo)記的PLGA納米粒,對其在圓窗膜的通透性及轉(zhuǎn)運機制進行研究。豚鼠,鼓室注射PLGA納米粒30 min后,分離出圓窗膜進行膜鋪片操作,采用激光掃描共聚焦顯微鏡觀察,發(fā)現(xiàn)香豆素-6的綠色熒光在圓窗膜中廣泛分布,說明納米粒可以進入圓窗膜;同時采集納米粒給藥后的外淋巴樣本進行透射電鏡觀察發(fā)現(xiàn)納米�？梢砸酝暾问竭M入外淋巴,說明了納米粒鼓室給藥后,可以穿越圓窗膜到達外淋巴空間。通過改變鼓室注射PLGA納米粒的濃度,選用了0.01 g/ml,0.03 g/ml及0.09 g/ml三個濃度點對納米粒穿越圓窗膜進行濃度依賴性考察,結(jié)果發(fā)現(xiàn)圓窗膜內(nèi)的熒光量隨著給藥濃度的增加而增加。選用了10 min,30 min及60 min三個時間點,對PLGA納米粒穿過圓窗膜進行時間依賴性考察,發(fā)現(xiàn)在給藥30 min內(nèi),圓窗膜內(nèi)的熒光量隨著時間的增加而增加,但是在30 min后,隨著時間的增加而降低。對鼓室注射PLGA納米粒30 min的豚鼠圓窗膜進行透射電鏡觀察,與正常組的圓窗膜相比,給藥組的圓窗膜中發(fā)現(xiàn)了大量的內(nèi)吞及胞吐小泡,同時圓窗膜外上皮細胞之間的緊密連接并沒有打開,因此可知PLGA跨越圓窗膜主要是通過內(nèi)吞的方式進入細胞,在細胞內(nèi)進行囊泡轉(zhuǎn)運,而不是通過細胞旁路途經(jīng)。采用抑制不同內(nèi)吞途徑的抑制劑,發(fā)現(xiàn)PLGA納米粒主要是以小窩蛋白以及巨胞飲介導(dǎo)的內(nèi)吞途徑進入細胞。對細胞內(nèi)的溶酶體進行熒光標(biāo)記,采用激光掃描共聚焦共定位技術(shù),對溶酶體及香豆素-6標(biāo)記的PLGA納米粒進行共定位,發(fā)現(xiàn)納米粒攝取進入細胞后,會進入細胞的溶酶體。采用與高爾基體相關(guān)的抑制劑發(fā)現(xiàn)細胞內(nèi)的內(nèi)質(zhì)網(wǎng)及高爾基體可以介導(dǎo)納米粒的胞吐。對內(nèi)耳疾病進行合理有效的治療很大程度上取決于耳蝸內(nèi)恒定的藥物濃度。殼聚糖(Chitosan,CS)是一種生物可降解的聚合物,由于其粘附特性是一種優(yōu)良的藥物載體,可以增加納米粒與圓窗膜的接觸時間,同時殼聚糖具有打開上皮細胞間緊密連接的作用,可以增加納米粒的細胞旁路途經(jīng)。采用離子交聯(lián)法制備載香豆素-6的CS納米粒,鼓室給藥后進行圓窗膜鋪片,采用激光掃描共聚焦顯微鏡成像后得到的圓窗膜的3D重建圖像以及外淋巴的透射電鏡充分證明了殼聚糖納米�？梢源┻^圓窗膜進入外淋巴。對殼聚糖跨越圓窗膜進行時間依賴性考察,選取了5個給藥時間點,10 min、30 min、1 h、2 h及4 h,結(jié)果顯示在給藥60 min以前,圓窗膜中香豆素-6的熒光量隨著時間的延長而增加,但是在60 min到240 min的時間段內(nèi)逐漸減弱;通過改變殼聚糖納米粒給藥的濃度,選用了3個濃度點5 mg/ml,10 mg/ml及20 mg/ml,發(fā)現(xiàn)圓窗膜中殼聚糖納米粒的熒光量隨著給藥納米粒濃度的增加而增加,呈現(xiàn)濃度依賴性。納米粒進入上皮屏障有細胞內(nèi)和細胞間兩種途徑,采用透射電鏡對殼聚糖給藥后的圓窗膜進行成像,發(fā)現(xiàn)殼聚糖納米�？梢砸约毎月芳翱缂毎麅煞N方式進入圓窗膜。采用不同途徑的內(nèi)吞抑制劑處理圓窗膜發(fā)現(xiàn)殼聚糖納米粒以小窩蛋白,網(wǎng)格蛋白及巨胞飲三種途徑進入圓窗膜細胞,說明了殼聚糖納米粒與圓窗膜細胞之間的非特異性相互作用。采用熒光共定位技術(shù)觀察到了殼聚糖納米粒在溶酶體的分布,高爾基體相關(guān)的抑制劑說明殼聚糖納米�？梢栽趦�(nèi)質(zhì)網(wǎng)及高爾基體的介導(dǎo)下胞吐出細胞。綜上所述,本課題第一次直觀證明了PLGA納米粒及CS納米粒確實可以穿過圓窗膜到達外淋巴,并闡明了納米粒在圓窗膜的內(nèi)吞,細胞內(nèi)轉(zhuǎn)運及胞吐過程的詳細的機制,為制劑的進一步優(yōu)化提供指導(dǎo)。
[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.

【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943

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相關(guān)期刊論文 前1條

1 劉婭;孫建軍;孔維佳;江平;姜偉;;離體圓窗膜對地塞米松不同制劑的通透性[J];中華耳鼻咽喉頭頸外科雜志;2006年03期

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本文編號：1644576