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具有pH响应及细胞核靶向功能的聚合物胶束在癌症治疗中的研究

发布时间:2018-12-10 06:18
【摘要】:近年来,利用纳米载体对癌症进行靶向治疗,在抗癌治疗研究中受到了广泛的关注。为了提高化疗药物的抗癌效果以及减少其对系统的毒副作用,增加肿瘤细胞对纳米颗粒的摄取和实现细胞核药物传递显得尤为重要。考虑利用细胞穿透肽(CPP)修饰具有刺激响应性的纳米载体,不仅能达到肿瘤靶向的目的,还能促进肿瘤细胞大量摄取纳米颗粒,从而有效抑制癌细胞生长繁殖。在本论文中第二章,我们成功合成了具有细胞穿透肽兼细胞核定位信号Tat修饰的pH响应性聚合物DA-Tat-PEG-PCL(DA-Tat-PECL)。为了改善广谱抗癌药物10-羟基喜树碱(HCPT)的疏水性,提高载药量(LC)和载药效率(EE),我们把HCPT接枝在甲氧基聚乙二醇(mPEG)上,成功合成了改性药物mPEG-HCPT。我们通过核磁共振氢谱(1H NMR),凝胶渗透色谱(GPC),红外光谱(FT-IR)和高效液相色谱(HPLC)验证了共聚物结构的正确性。通过溶剂挥发法,我们制备出具有细胞核靶向和pH响应性的胶束PECL/DA-Tat-M,该胶束具有较低的临界胶束浓度(CMC),热力学稳定性良好,且具备核壳结构,能有效包载疏水性药物。纳米激光粒度仪DLS和透射电镜TEM的结果显示,该胶束呈均一球形,粒径在80nm左右。体外模拟药物释放实验结果表明:包载mPEG-HCPT的胶束的释药速率比包载HCPT的胶束的释药速率快,且胶束的LC和EE分别在5%和70%以上。胶束的pH响应性考察结果显示,在pH 6.8条件下,二甲基马来酸酐(DA)与Tat之间的酰胺键迅速断裂,Tat完全暴露,使胶束发生电荷反转,表面电荷由负变正,且粒径也增大到90 nm左右。在第三章中,对该电荷反转胶束PECL/DA-Tat-M进行了一系列体外生物学评价,结果表明PECL/DA-Tat-blank M空白胶束具有良好的生物相容性,载入mPEG-HCPT后能抑制肿瘤细胞,且在pH6.8时具有最低的IC50值。我们发现,肿瘤细胞对该胶束的的摄取,以及亚细胞定位均受到pH的影响,在pH 6.8时,该胶束被肿瘤细胞大量摄取,进入细胞后能有效富集在细胞核周围,药物释放后顺利进入细胞核,这些结果均表明,在弱酸性环境下,DA从共聚物上脱落,Tat暴露出来,促进了细胞的内吞作用,进入细胞后,Tat发挥细胞核定位的作用,使胶束实现细胞核靶向功能。在第四章,建立了 4T1小鼠乳腺癌原位模型和异位模型,用于验证该胶束的体内抗肿瘤效果的研究。结果显示与对照组,空白胶束组,HCPT组,mPEG-HCPT组合PECL-M胶束组相比,PECL/DA-Tat-M胶束不仅能显著抑制肿瘤生长,且能集中分布于肿瘤组织,避免全身性的副作用,提高了实验动物的生存率,有较高的安全性。肿瘤组织的TUNEL染色结果表明,该胶束组的肿瘤细胞凋亡率最高,有最佳抑瘤效果。对原位模型自发肺转移的研究结果也表明,PECL/DA-Tat-M胶束抑制了肺转移,肺部的HE染色分析显示该组小鼠肺泡结构正常,未发生肺转移现象,进一步证明了这种电荷反转胶束有效的抑制了自发的肺转移。
[Abstract]:In recent years, the use of nano-carrier targeted cancer therapy, cancer treatment research has received extensive attention. In order to improve the anticancer effect of chemotherapeutic drugs and reduce the toxicity and side effects of chemotherapeutic drugs, it is particularly important to increase the uptake of nanoparticles by tumor cells and to achieve nuclear drug delivery. It is considered that the modification of the stimulation-responsive nano-carrier by cell penetrating peptide (CPP) can not only achieve the goal of tumor targeting, but also promote tumor cells to absorb a large number of nanoparticles, thus effectively inhibit the growth and reproduction of cancer cells. In chapter 2, we successfully synthesized pH responsive polymer DA-Tat-PEG-PCL (DA-Tat-PECL) with cellular penetrating peptide and nuclear localization signal modified by Tat. In order to improve the hydrophobicity of 10-hydroxycamptothecin (HCPT), increase the drug loading capacity (LC) and drug loading efficiency (EE), we grafted HCPT onto methoxy polyethylene glycol (mPEG) and synthesized the modified drug mPEG-HCPT. successfully. The structure of copolymers was verified by 1H NMR), gel permeation chromatography (FT-IR) and high performance liquid chromatography (HPLC). A micelle PECL/DA-Tat-M, with nuclear targeting and pH responsiveness was prepared by solvent volatilization. The micelle has a low critical micelle concentration, (CMC), has good thermodynamic stability and has a core-shell structure. Can effectively package hydrophobic drugs. The results of DLS and TEM show that the micelle is homogeneous spherical and its particle size is about 80nm. The drug release rate of micelles coated with mPEG-HCPT was faster than that of micelles coated with HCPT in vitro, and the LC and EE of micelles were more than 5% and 70%, respectively. The pH response of the micelles showed that under pH 6.8, the amide-bond between (DA) and Tat broke rapidly, and Tat was completely exposed, which caused the micelle to reverse the charge and the surface charge changed from negative to positive. The particle size also increased to about 90 nm. In chapter 3, a series of biological evaluation of the charge reversal micelle PECL/DA-Tat-M in vitro was carried out. The results showed that the blank micelles of PECL/DA-Tat-blank M had good biocompatibility and could inhibit tumor cells after loading mPEG-HCPT. And has the lowest IC50 value in pH6.8. We found that the uptake and subcellular localization of the micelles by tumor cells were affected by pH. At pH 6.8, the micelles were ingested in large quantities by tumor cells and could be effectively enriched around the nucleus after entering the cells. The results showed that DA shedded from the copolymer and Tat was exposed, which promoted the endocytosis of the cells. After entering the cells, Tat played the role of nuclear localization. The micelles can achieve nuclear targeting function. In chapter 4, an in situ model and an ectopic model of breast cancer in 4T1 mice were established to verify the antitumor effect of the micelle in vivo. The results showed that compared with control group, blank micelle group, HCPT group and mPEG-HCPT combined PECL-M micelle group, PECL/DA-Tat-M micelles could not only significantly inhibit tumor growth, but also concentrate on tumor tissue and avoid systemic side effects. The survival rate of experimental animals was improved and the safety was higher. The results of TUNEL staining showed that the micelle group had the highest apoptosis rate and the best tumor inhibition effect. The results of spontaneous lung metastasis in situ model also showed that PECL/DA-Tat-M micelles inhibited lung metastasis, and the lung HE staining showed that the alveolar structure of the group was normal and no pulmonary metastasis occurred. It is further demonstrated that this charge reversal micelle effectively inhibits spontaneous lung metastasis.
【学位授予单位】:西南交通大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R730.5

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