本文關(guān)鍵詞： 多肽 乳酸-羥基乙酸共聚物 紫杉醇 納米粒 靶向性　出處：《昆明醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]：乳腺癌是女性最嚴(yán)重的危害性疾病,治療中將藥物直接作用于病變靶點能夠有效提高治療作用。本實驗室前期發(fā)現(xiàn)了特異性作用于乳腺癌MDA-MB-231細(xì)胞的多肽,命名為PI�，F(xiàn)我們將PI多肽連接于載紫杉醇PLGA納米粒上,使之靶向性作用于腫瘤細(xì)胞,同時評估其在體內(nèi)外對乳腺癌MDA-MB-231細(xì)胞的療效。[方法]：采用乳化-溶劑揮發(fā)技術(shù)制作包載有紫杉醇的PLGA生物納米粒(PTX-NPs)。將異硫氰酸熒光素(FITC)標(biāo)記的多肽PI (MDA-MB-231乳腺癌細(xì)胞特異轉(zhuǎn)導(dǎo)肽)與PTX-NPs共同置于EDC/NHS交聯(lián)體系中,進(jìn)行化學(xué)連接,建立PI修飾的載藥納米粒(PPTX-NPs)。選香豆素-6作為熒光探針,以相同的方法制得香豆素-6-NPs,以異硫氰酸熒光素標(biāo)記的多肽TAT制備TAT修飾的載藥納米粒(TPTX-NPs)。對納米粒進(jìn)行理化性質(zhì)評估：掃描電鏡(SEM)觀察納米粒表面形態(tài)及大��；高效液相色譜儀(HPLC)測定納米粒載藥量和體外釋放規(guī)律：傅立葉紅外光譜(FTIR)驗證PI與PTX-NPs的連接。納米粒細(xì)胞毒性實驗：培養(yǎng)MDA-MB-231細(xì)胞和TCA-8113細(xì)胞,將兩株細(xì)胞分別與香豆素-6-NPs(空白對照組)、TPTX-NPs(陽性對照組)、PPTX-NPs(實驗組)共培養(yǎng),分別在12h和24h時熒光顯微鏡下觀察細(xì)胞對不同納米粒的攝取強弱。MTS比色法觀察PTX組、PTX-NPs組、PPTX-NPs組、TPTX-NPs組對兩株細(xì)胞的細(xì)胞毒性作用。納米粒在小鼠體內(nèi)的代謝及抑瘤作用：飼養(yǎng)昆明(KM)小鼠,由尾靜脈注射給藥,分隔不同時間段心臟取血,觀察小鼠體內(nèi)血藥動力學(xué)特征。在Balb/c-nu裸鼠上構(gòu)建MDA-MB-231乳腺癌模型,以生理鹽水為對照組,比較PTX注射液、PTX-NPs懸液、PPTX-NPs懸液對腫瘤的抑制作用。[結(jié)果]：成功構(gòu)建了PI修飾的載藥納米粒,掃描電鏡可見載藥納米粒及多肽修飾的納米粒比空白納米粒略有增大,且多肽修飾的納米粒出現(xiàn)了部分顆粒團(tuán)聚現(xiàn)象。經(jīng)HPLC測得PI多肽修飾的載藥納米粒載藥量為8.4±0.59%,藥物體外釋放前兩天累積為32%,到達(dá)第12天時累積釋放量為58%。熒光顯微鏡檢測MDA-MB-231細(xì)胞對PPTX-NPs和TPTX-NPs攝取率高,在12h和24h時對前者攝取率分別約為80%和95%以上,對后者攝取率分別約為60%和75%,而TCA-8113細(xì)胞對TPTX-NPs的攝取率12h和24h分別約57%和80%,而對PPTX-NPs幾乎不攝取。MTS試驗結(jié)果顯示,PPTX-NPs對MDA-MB-231細(xì)胞的殺傷性優(yōu)于PTX-NPs組,在作用24h時細(xì)胞存活率分別為56.04±0.86%和60.65±0.49%,作用至48h時細(xì)胞存活率分別為34.80±0.60%及45.50±0.81%(P0.05)。納米粒作用于小鼠體內(nèi)藥動學(xué)結(jié)果PPTX-NPs懸液的T1/2是PTX注射液的2.1倍,AUC0→24是PTX注射液的4.24倍,兩者的AUC0-24h及T1/2參數(shù)均有統(tǒng)計學(xué)差異(P0.05)。PPTX-NPs懸液組治療荷MDA-MB-231裸鼠后對腫塊生長抑制率為85.5%,高于PTX注射液組和PTX-NPs懸液組(P0.05)。[結(jié)論]：乳腺癌特異轉(zhuǎn)導(dǎo)多肽(PI)修飾的載藥納米粒具有良好的靶向性和安全性,能促進(jìn)所載藥物進(jìn)入腫瘤細(xì)胞,增強藥物對MDA-MB-231細(xì)胞的靶向殺傷性。
[Abstract]:[objective]: breast cancer is the most serious disease in women. The direct action of drugs on the target of breast cancer can effectively improve the therapeutic effect. The polypeptides specific to breast cancer MDA-MB-231 cells have been found in our laboratory. We now attach Pi peptide to paclitaxel loaded PLGA nanoparticles to target tumor cells. At the same time, the therapeutic effect on breast cancer MDA-MB-231 cells in vitro and in vivo was evaluated. [methods] PLGA bionanoparticles containing paclitaxel containing paclitaxel were prepared by emulsification-solvent volatilization technique. The polypeptide Pi MDA-MB-231 was labeled with fluorescein isothiocyanate (FITC). The cell-specific transduction peptide was co-located with PTX-NPs in EDC/NHS crosslinking system. The PPTX-NPsNPs were chemically linked to the PPTX-NPs, and coumarin -6 was selected as the fluorescent probe. Coumarin -6-NPs were prepared by the same method and TAT modified drug loaded nanoparticles (TPTX-NPs) were prepared by using fluorescein isothiocyanate labeled polypeptide TAT. The physicochemical properties of the nanoparticles were evaluated: scanning electron microscopy (SEM) was used to observe the surface morphology and size of the nanoparticles. High performance liquid chromatograph (HPLC) for the determination of drug loading and in vitro release of nanoparticles: Fourier transform infrared spectroscopy (FTIR) was used to verify the connection between Pi and PTX-NPs. The two cells were co-cultured with coumarin-6-NPs (TPTX-NPs). Cell uptake of different nanoparticles was observed under fluorescence microscope for 12 h and 24 h respectively. MTS colorimetric method was used to observe the cytotoxicity of PTX group and PPTX-NPs group to two cell lines. The metabolism of nanoparticles in mice and the inhibition of tumor activity were observed. KM) mice, MDA-MB-231 breast cancer model was established on Balb/c-nu nude mice, and normal saline was used as control group. The inhibitory effect of PPTX-NPs suspension of PTX injection on tumor was compared. [results]: Pi modified drug loaded nanoparticles were successfully constructed. Scanning electron microscope showed that drug loaded nanoparticles and polypeptide modified nanoparticles were slightly larger than that of blank nanoparticles. The drug loading capacity of Pi polypeptide modified nanoparticles was 8.4 鹵0.59 by HPLC, the cumulative amount was 32 before release in vitro, and the cumulative release amount was 58g at 12th day after release in vitro, and the fluorescence showed that the concentration of Pi polypeptide modified nanoparticles was 8. 5 鹵0. 59, and that of Pi polypeptide modified nanoparticles was 8. 4 鹵0. 59, 2 days before release in vitro, the cumulative release amount was 58%. The uptake rate of PPTX-NPs and TPTX-NPs in MDA-MB-231 cells was high by micromicroscopy. At 12 h and 24 h, the uptake rates of the former were above 80% and 95%, respectively. For the latter, the uptake rates of TPTX-NPs were about 60% and 75, respectively, while the uptake rates of TPTX-NPs in TCA-8113 cells were about 57% and 80 at 12 h and 24 h, respectively. The results of almost no uptake. MTS test showed that PPTX-NPs were more lethal to MDA-MB-231 cells than to PTX-NPs cells. The cell survival rate was 56.04 鹵0.86% and 60.65 鹵0.49respectively at 24h and 34.80 鹵0.60% and 45.50 鹵0.85 at 48h, respectively. The pharmacokinetic results showed that the T1 / 2 of PPTX-NPs suspension was 2.1-fold of that of PTX injection. 鈫，

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