本文選題：PEG-PLL-PLGA + 納米顆粒��； 參考：《東南大學(xué)》2016年博士論文

【摘要】：第一部分PEG-PLL-PLGA聚合物納米粒的生物相容性以及安全性研究目的本部分實驗研究評估聚乙二醇(PEG)-聚賴氨酸(PLL)-聚乳酸羥基乙酸(PLGA) (PEG-PLL-PLGA)納米粒(nanoparticles, NPs)的生物相容性及安全性。方法選用可生物降解材料PEG、PLL及PLGA作為合成新型納米載藥系統(tǒng)(nano drug delivery system, NDDS)的原料,通過優(yōu)化反應(yīng)條件,制得PEG-PLL-PLGA聚合物納米粒,并將PEG-PLL-PLGA包載經(jīng)典化療藥物柔紅霉素(doxorubicin,DNR)。采用一系列實驗檢測該納米材料的生物相容性,如MTT實驗評價PEG-PLL-PLGA對小鼠成纖維細(xì)胞L929的細(xì)胞毒性；流式細(xì)胞術(shù)(flow cytometry,FCM)檢測細(xì)胞內(nèi)DNR濃度和細(xì)胞凋亡率；溶血試驗評價納米粒有無溶血作用；小鼠尾靜脈注射DNR-PEG-PLL-PLGA以測定其半數(shù)致死量(median lethal dose,LD50)；急性毒性實驗采用小鼠眼球取血方法,檢測小鼠肝腎功能；病理組織學(xué)蘇木素-伊紅(haematoxylin-eosin, HE)染色檢測小鼠主要臟器的病理變化；微核試驗(micronucleus, MN)評價其有無致畸及致突變作用。結(jié)果成功制備PEG-PLL-PLGA聚合物納米粒,透射電鏡(transmission electron microscope, TEM)下PEG-PLL-PLGA呈球形或近似球形,表面光滑,平均粒徑為229+15 nm,多分散系數(shù)(polydispersity index, PI)為0.085±0.017(n= 3), Zeta電位為-20.1±1.6 mV；不同濃度的PEG-PLL-PLGA納米粒的細(xì)胞毒性均為1級,均屬對細(xì)胞無毒性范疇；FCM顯示DNR組的相對熒光強(qiáng)度(relative fluorescence intensity, RFI)為4.27+2.06, DNR-PEG-PLL-PLGA組RFI為4.39±1.89,與空白對照組相比,DNR組和DNR-PEG-PLL-PLGA組均能顯著增加細(xì)胞內(nèi)DNR濃度(P 0.05), PEG-PLL-PLGA實驗組與對照組相比,凋亡率沒有顯著差異(P0.05)；肉眼觀察法顯示無溶血發(fā)生；LDso為464.4 mg/kg,95%的可信區(qū)間為399-541.8 mg/kg,納米粒具有較廣的安全值范圍；組織病理學(xué)HE染色檢測示,DNR-PEG-PLL-PLGA實驗組與對照組相比,沒有顯著的組織結(jié)構(gòu)和細(xì)胞的病理變化形態(tài)；微核試驗證實該納米材料無致畸或致突變作用。結(jié)論PEG-PLL-PLGA在體外和體內(nèi)均無毒性,具有良好的生物相容性,同時為PEG-PLL-PLGA作為化療藥物傳輸系統(tǒng)的載體奠定了基礎(chǔ)。第二部分PEG-PLL-PLGA聚合物納米粒的靶向逆轉(zhuǎn)腫瘤多藥耐藥研究目的本部分實驗研究轉(zhuǎn)鐵蛋白(transferrin, Tf)修飾的PEG-PLL-PLGA納米粒共聚合抗腫瘤藥物DNR和漢防己甲素(Tetrandrine, Tet)治療白血病裸鼠移植瘤多藥耐藥(multidrug resistance, MDR)的療效及體內(nèi)靶向性分布。方法選用復(fù)乳化溶劑揮發(fā)法制備共載DNR和Tet的PEG-PLL-PLGA,利用載體末端羥基二咪唑(carbonyldiimidazole, CDI)基團(tuán)能自發(fā)地與Tf的氨基反應(yīng),形成Tf-PEG-PLL-PLGA,采用高速冷凍離心法及凝膠滲透色譜法檢測蛋白交聯(lián)率,合成DNR-Tet-Tf-PEG-PLL-PLGA (PEG-PLL-PLGA縮寫成NPs,即DNR-Tet-Tf-NPs)聚合物載藥納米粒。建立白血病耐藥細(xì)胞株K562/A02裸鼠皮下移植瘤模型,當(dāng)腫瘤長大至75-150 mm3時,隨機(jī)分為6組：生理鹽水對照組,DNR組,DNR和Tet組,DNR-NPs組,DNR-Tet-NPs組和DNR-Tet-Tf-NPs組,每組6只,采用尾靜脈注射方式,隔天給藥,觀察納米粒的抗腫瘤作用,并測定腫瘤的生長體積,計算腫瘤抑瘤率。用藥14天后處死動物,取各組心臟、肝臟、脾臟、肺臟、腎臟及腫瘤組織行病理學(xué)觀察。免疫組化檢測腫瘤組織中凋亡相關(guān)基因Caspase-3, Bax, Bcl-2和Survivin的表達(dá)。使用實時熒光定量PCR(quantitative real-time polymerase chain reaction, qRT-PCR)以及免疫蛋白印跡試驗(Western blot)檢測腫瘤靶向及耐藥相關(guān)抗體TfR, P-gp, MRP和NF-κB的mRNA及蛋白的表達(dá)。高效液相色譜法(high performance liquid chromatography, HPLC)檢測DNR在血漿、腫瘤組織和主要臟器的濃度。FCM檢測近紅外染料(near-infrared fluorescent, NIRF) NIR797是否包載至納米粒中,近紅外活體成像實驗檢測NIR797-labeled DNR-NPs和NIR797-labeled DNR-Tf-NPs在裸鼠體內(nèi)的分布情況。結(jié)果DNR-Tet-Tf-NPs組腫瘤抑制率最高(P0.05),抑瘤作用最強(qiáng),同時能夠逆轉(zhuǎn)腫瘤MDR,且具有靶向抗腫瘤作用。HE染色顯示各組心臟、肝臟、脾臟、肺臟、腎臟及腫瘤組織無明顯病理改變。腫瘤組織免疫組化觀察顯示,與對照組和DNR組相比,Bax和Caspase-3蛋白在DNR-Tet-NPs組和DNR-Tet-Tf-NPs組中表達(dá)明顯增加,而Bcl-2和Survivin蛋白表達(dá)明顯減少,并且在DNR-Tet-Tf-NPs組中Bax和Caspase-3蛋白表達(dá)最強(qiáng),Bcl-2和Survivin蛋白表達(dá)最弱。實時定量PCR和Western blot檢測結(jié)果示DNR-Tet-Tf-NPs組能夠下調(diào)P-gp, MRP, NF-κB的mRNA及蛋白的表達(dá),上調(diào)TfR的mRNA及蛋白的表達(dá)。HPLC實驗結(jié)果顯示DNR-Tet-Tf-NPs組經(jīng)裸鼠尾靜脈給藥4h后,在腫瘤組織中的DNR濃度明顯高于DNR-Tet-NPs組。近紅外活體成像結(jié)果顯示,通過主要臟器及腫瘤部位的近紅外成像發(fā)現(xiàn),大量的熒光信號主要聚集在腫瘤部位。在注射72 h后,最強(qiáng)的信號到達(dá)了裸鼠成瘤的部位。非Tf修飾的NIR797-labeled DNR-NPs組也有相同的成像的結(jié)果,但是腫瘤的熒光信號比Tf修飾的NIR797-labeled DNR-Tf-NPs組弱。結(jié)論DNR-Tet-Tf-NPs具有良好的逆轉(zhuǎn)耐藥及主動靶向能力,為腫瘤的靶向治療提供了一條新途徑。
[Abstract]:The biocompatibility and safety of PEG - PLL - PLGA nanoparticles ( PEG - PLL - PLGA ) nanoparticles ( PEG - PLL - PLGA ) nanoparticles ( PEG - PLL - PLGA ) nanoparticles were evaluated . The biocompatibility of the nano - material was tested by a series of experiments , such as MTT assay to evaluate the cytotoxicity of PEG - PLL - PLGA on mouse fibroblast L929 ;
Flow cytometry ( FCM ) was used to detect the concentration and cell apoptosis rate in the cells .
Hemolysis test was used to evaluate the hemolytic effect of nanoparticles .
The median lethal dose ( LD 50 ) was determined by intravenous injection of dnr - PEG - PLL - PLGA in tail vein of mice .
In acute toxicity experiment , mouse eyeball blood taking method was used to detect the liver and kidney function of mice .
The pathological changes of the main organs of mice were detected by pathological histology and eosin ( HE ) staining .
The results showed that PEG - PLL - PLGA nanoparticles were spherical or approximately spherical in PEG - PLL - PLGA polymer nanoparticles with smooth surface , average particle size of 229 + 15 nm , polydispersity index ( PI ) of 0.085 鹵 0.17 ( n = 3 ) , zeta potential of - 20.1 鹵 1.6 mV .
The cytotoxicity of PEG - PLL - PLGA nanoparticles with different concentrations was grade 1 , which belongs to the non - toxic category of cells .
The relative fluorescence intensity ( rfi ) was 4.27 + 2.06 , the rfi of rfi in dnr group was 4.27 + 2.06 , rfi of dnr - peg - pll - PLGA group was 4.39 鹵 1.89 . Compared with the control group , there was no significant difference in the apoptosis rate ( P0.05 ) .
naked eye observation showed no hemolysis ;
LDso is 464.4 mg / kg , 95 % confidence interval is 399 - 541.8 mg / kg , and the nanoparticles have a wide range of safety values ;
Tissue pathology HE staining showed that there was no significant difference in structure and pathological changes compared with the control group .
In vitro and in vivo , PEG - PLL - PLGA nanoparticles ( PEG - PLL - PLGA ) modified with PEG - PLL - PLGA nanoparticles were used to study the antitumor effect of tumor . The expression of P - gp , MRP and NF - 魏B mRNA and protein in nude mice was detected by high performance liquid chromatography ( HPLC ) .
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.53

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 歐陽松應(yīng),楊冬,歐陽紅生,馬鶴雯;實時熒光定量PCR技術(shù)及其應(yīng)用[J];生命的化學(xué);2004年01期

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本文編號：2031561