乳腺癌特异性转导多肽修饰的载药纳米粒的抗肿瘤效应研究

发布时间：2018-02-19 18:07

  本文关键词： 多肽 乳酸-羟基乙酸共聚物 紫杉醇 纳米粒 靶向性　出处：《昆明医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：[目的]：乳腺癌是女性最严重的危害性疾病,治疗中将药物直接作用于病变靶点能够有效提高治疗作用。本实验室前期发现了特异性作用于乳腺癌MDA-MB-231细胞的多肽,命名为PI。现我们将PI多肽连接于载紫杉醇PLGA纳米粒上,使之靶向性作用于肿瘤细胞,同时评估其在体内外对乳腺癌MDA-MB-231细胞的疗效。[方法]：采用乳化-溶剂挥发技术制作包载有紫杉醇的PLGA生物纳米粒(PTX-NPs)。将异硫氰酸荧光素(FITC)标记的多肽PI (MDA-MB-231乳腺癌细胞特异转导肽)与PTX-NPs共同置于EDC/NHS交联体系中,进行化学连接,建立PI修饰的载药纳米粒(PPTX-NPs)。选香豆素-6作为荧光探针,以相同的方法制得香豆素-6-NPs,以异硫氰酸荧光素标记的多肽TAT制备TAT修饰的载药纳米粒(TPTX-NPs)。对纳米粒进行理化性质评估：扫描电镜(SEM)观察纳米粒表面形态及大小；高效液相色谱仪(HPLC)测定纳米粒载药量和体外释放规律：傅立叶红外光谱(FTIR)验证PI与PTX-NPs的连接。纳米粒细胞毒性实验：培养MDA-MB-231细胞和TCA-8113细胞,将两株细胞分别与香豆素-6-NPs(空白对照组)、TPTX-NPs(阳性对照组)、PPTX-NPs(实验组)共培养,分别在12h和24h时荧光显微镜下观察细胞对不同纳米粒的摄取强弱。MTS比色法观察PTX组、PTX-NPs组、PPTX-NPs组、TPTX-NPs组对两株细胞的细胞毒性作用。纳米粒在小鼠体内的代谢及抑瘤作用：饲养昆明(KM)小鼠,由尾静脉注射给药,分隔不同时间段心脏取血,观察小鼠体内血药动力学特征。在Balb/c-nu裸鼠上构建MDA-MB-231乳腺癌模型,以生理盐水为对照组,比较PTX注射液、PTX-NPs悬液、PPTX-NPs悬液对肿瘤的抑制作用。[结果]：成功构建了PI修饰的载药纳米粒,扫描电镜可见载药纳米粒及多肽修饰的纳米粒比空白纳米粒略有增大,且多肽修饰的纳米粒出现了部分颗粒团聚现象。经HPLC测得PI多肽修饰的载药纳米粒载药量为8.4±0.59%,药物体外释放前两天累积为32%,到达第12天时累积释放量为58%。荧光显微镜检测MDA-MB-231细胞对PPTX-NPs和TPTX-NPs摄取率高,在12h和24h时对前者摄取率分别约为80%和95%以上,对后者摄取率分别约为60%和75%,而TCA-8113细胞对TPTX-NPs的摄取率12h和24h分别约57%和80%,而对PPTX-NPs几乎不摄取。MTS试验结果显示,PPTX-NPs对MDA-MB-231细胞的杀伤性优于PTX-NPs组,在作用24h时细胞存活率分别为56.04±0.86%和60.65±0.49%,作用至48h时细胞存活率分别为34.80±0.60%及45.50±0.81%(P0.05)。纳米粒作用于小鼠体内药动学结果PPTX-NPs悬液的T1/2是PTX注射液的2.1倍,AUC0→24是PTX注射液的4.24倍,两者的AUC0-24h及T1/2参数均有统计学差异(P0.05)。PPTX-NPs悬液组治疗荷MDA-MB-231裸鼠后对肿块生长抑制率为85.5%,高于PTX注射液组和PTX-NPs悬液组(P0.05)。[结论]：乳腺癌特异转导多肽(PI)修饰的载药纳米粒具有良好的靶向性和安全性,能促进所载药物进入肿瘤细胞,增强药物对MDA-MB-231细胞的靶向杀伤性。
[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 卤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 卤0. 59, and that of Pi polypeptide modified nanoparticles was 8. 4 卤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 卤0.86% and 60.65 卤0.49respectively at 24h and 34.80 卤0.60% and 45.50 卤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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