基于DNA四面体的级联式药物递送系统逆转乳腺癌细胞耐药研究

发布时间：2018-08-05 12:11

【摘要】：恶性肿瘤严重威胁人类生命健康,目前为止,化疗仍是治疗恶性肿瘤的主要手段,但传统的化疗药物易产生耐药,成为化疗的主要问题之一。此外,化疗药物还存在肿瘤靶向性差、毒副作用大等缺点。因此,构建能够克服恶性肿瘤耐药并提高肿瘤靶向性、降低化疗药物毒性的一体化药物递送系统(Drug Delivery Systems,DDSs)迫在眉睫。DNA纳米技术,作为纳米材料科学的一种新技术,自问世以来,由于其可设计性和可预测性,且可形成独特的形貌特征而备受关注。其中,DNA四面体(DNA Tetrahedron),具有结构与性能稳定、易于多样化修饰等优点,被认为是一种理想的药物载体。脂质体(Liposome)具有纳米尺度、良好的生物相容性及高度的生物安全性等特征,近年来作为化疗药物的转运载体被广泛应用于靶向药物递送领域。细胞核为多数化疗药物的主要靶点,但肿瘤细胞对化疗药物的外排作用导致多数化疗药物难以到达细胞核发挥作用。细胞核核孔直径约为9 nm,而自组装的DNA四面体粒径约为5 nm,能够直接通过核孔高效地进入细胞核中,基于此,本研究首先将临床一线化疗药物阿霉素(Doxorubicin,DOX)镶嵌入DNA四面体(Td)中形成DOX@Td;然后,使用免疫脂质体将DOX@Td高效包裹,构建基于DNA四面体的脂质体(DOX@Td@Lipo)高效核转运纳米药物递送系统。本研究所构建的药物递送系统的特征为:一、利用免疫脂质体将DOX@DNA四面体偶联体高效地转运至肿瘤细胞内;二、利用DNA四面体小尺寸(小于细胞核孔)及核内可降解特性,将DOX高效的转运至细胞核中,有效地避免了肿瘤细胞的药物外排途径,逆转肿瘤细胞耐药。该系统显著地提高了DOX对耐药乳腺癌的治疗效率,并降低了DOX的毒副作用。然后,本研究以人类乳腺癌细胞MCF-7和人类耐药乳腺癌细胞MCF-7/ADR为细胞模型以及以MCF-7/ADR荷瘤BLAB/C裸鼠为动物模型,主要进行了以下三方面的研究:1.DOX@Td@Lipo级联式药物递送系统的制备及表征:单链DNA(ssDNA)复性并碱基互补配对自组装成DNA四面体,原子力显微镜(Automatic Force Microscope,AFM)以及凝胶成像表征验证,结果显示制备四面体粒径在5 nm左右,且分布均匀;利用DOX易于插入到DNA双螺旋结构的性质,制备DOX@Td偶联物,通过电泳实验考察其稳定性,结果表明偶联DOX后的DNA四面体仍维持其四面体结构,具有较强稳定性;最终,采用逆向蒸发法制备DOX@Td@Lipo,用透射电子显微镜(Transmission Electron Microscope,TEM)对其表征,粒径在147 nm左右,并且粒径分布均匀。2.DOX@Td@Lipo级联式药物递送系统体外抗肿瘤活性及逆转肿瘤细胞耐药研究:系统考察DOX@Td@Lipo对MCF-7及MCF-7/ADR细胞的抗肿瘤活性,结果表明对于MCF-7细胞,DOX、DOX@Lipo以及DOX@Td@Lipo对MCF-7细胞的增殖抑制作用无显著性差别(48 h时抑制率分别为70.65%、69.55%、65.08%)。然而,DOX和DOX@Lipo组对ADR细胞的增殖抑制作用显著低于DOX@Td@Lipo组(48h时抑制率分别为21.23%、25.73%、79.83%)。表明在细胞水平,DOX@Td@Lipo成功地逆转了MCF-7/ADR细胞对DOX的耐药性进而提高了DOX杀伤肿瘤细胞的能力。此外,本研究还通过荧光定位的方法对所构建系统的细胞核靶向能力进行验证。结果显示,与DOX组相比,Td-FAM组、DOX@Td组及DOX@Td@Lipo组在MCF-7/ADR细胞核部位观测到FAM的绿色荧光和DOX的红色荧光,而DOX组和DOX@Lipo组在细胞核部位没有红色荧光出现。说明DOX不能进入耐药细胞细胞核,DOX@Lipo也不能将DOX运送入耐药细胞的细胞核,但是DOX@Td@Lipo药物递送系统能够将DOX转运入耐药细胞并且释放的DOX能够进入耐药细胞细胞核。3.DOX@Td@Lipo级联式药物递送系统体内抗肿瘤活性评价:以MCF-7/ADR荷瘤裸鼠为模型,评价所构建的药物递送系统的体内抗肿瘤活性。通过近红外光活体成像技术考察所构建的递送系统在荷瘤裸鼠体内的分布行为,结果显示,静脉注射后1 h后DOX@Td@Lipo即可在肿瘤组织富集,且随时间延长富集在肿瘤组织的药物越多。此外,结果还表明,免疫脂质体的包封增强了药物在裸鼠体内的循环时间。药效学结果显示,经过2周治疗,制剂DOX@Td@Lipo组对肿瘤的抑制率为66.7%,DOX@Lipo组抑制率41.67%,Td@Lipo组无治疗作用。说明所构建的药物递送系统对乳腺癌耐药肿瘤具有显著的治疗作用。体内抗肿瘤活性也通过病理学进行了进一步确证,通过对比正常组织器官发现,DOX@Td@Lipo显著降低了DOX的毒副作用。
[Abstract]:Malignant tumor is a serious threat to human life and health. Up to now, chemotherapy is still the main means to treat malignant tumors. However, traditional chemotherapeutic drugs are easy to produce drug resistance and become one of the main problems of chemotherapy. In addition, chemotherapy drugs still have the disadvantages of poor tumor targeting and toxic side effects. Drug Delivery Systems (DDSs), an integrated drug delivery system (DDSs), which is targeted for high tumor targeting, reduces the toxicity of chemotherapeutic drugs (DDSs), is an imminent.DNA nanotechnology. As a new technology of nano material science, since it was asked, it has attracted much attention because of its design and predictability and the formation of unique morphologies. DNA tetrahedron (DNA) Tetrahedron), with the advantages of stable structure and performance, easy to diversify and so on, it is considered as an ideal drug carrier. Liposome (Liposome) has the characteristics of nanoscale, good biocompatibility and high biological safety. In recent years, as a transport carrier for chemotherapeutic drugs, liposomes have been widely used in the field of targeting drug delivery. The nucleus is the main target of most chemotherapeutic drugs, but the efflux of the tumor cells to chemotherapeutic drugs causes most chemotherapy drugs to be difficult to reach the nucleus. The nuclear pore diameter of the nucleus is about 9 nm, and the self assembled DNA tetrahedron particle size is about 5 nm, which can directly enter the nucleus through the nuclear pore. Based on this, this study is based on this study. Doxorubicin (DOX) is inlaid into DNA tetrahedron (Td) to form DOX@Td, and then, the efficient nuclear transport system of DOX@Td@Lipo based on DNA tetrahedron (DOX@Td@Lipo) is constructed by using immune liposomes to encapsulate DOX@Td efficiently. The characteristics of the drug delivery system constructed in this study are the use of the immune system. Phytophthora transtransport DOX@DNA tetrahedron into tumor cells efficiently. Two, using small size of DNA tetrahedron (smaller than nuclear pore) and biodegradability in the nucleus, the efficient transport of DOX into the nucleus can effectively avoid the drug delivery pathway of tumor cells and reverse the drug resistance of tumor cells. This system significantly improves the DOX pair The treatment efficiency of drug-resistant breast cancer and the toxic and side effects of DOX were reduced. Then, this study took human breast cancer cell MCF-7 and human breast cancer cell MCF-7/ADR as the cell model and MCF-7/ADR tumor bearing BLAB/C nude mice as animal models. The following three aspects were mainly studied: the system of 1.DOX@Td@Lipo cascade drug delivery system Preparation and characterization: single strand DNA (ssDNA) complex and base pair complementary pairs are self assembled into DNA tetrahedron, atomic force microscopy (Automatic Force Microscope, AFM) and gel imaging characterization. The results show that the size of tetrahedron is about 5 nm, and the distribution is uniform, and DOX is easily inserted into the properties of DNA double helix structure, and DOX@Td coupling is prepared. The stability was investigated by electrophoretic test. The results showed that DNA tetrahedron after coupling DOX still maintained its tetrahedral structure and had strong stability. Finally, DOX@Td@Lipo was prepared by reverse evaporation method and characterized by transmission electron microscope (Transmission Electron Microscope, TEM). The particle size was about 147 nm, and the particle size distribution was uniform.2.DOX. @Td@Lipo cascade drug delivery system in vitro antitumor activity and reversal of tumor cell resistance: a systematic investigation of the antitumor activity of DOX@Td@Lipo on MCF-7 and MCF-7/ADR cells showed that there was no significant difference in the inhibitory effect of DOX, DOX@Lipo and DOX@Td@Lipo on the proliferation of MCF-7 cells (the inhibition rate of 48 h was 70., respectively. 65%, 69.55%, 65.08%). However, the inhibitory effect of DOX and DOX@Lipo on the proliferation of ADR cells was significantly lower than that in the DOX@Td@Lipo group (48h inhibition rate was 21.23%, 25.73%, 79.83%). It showed that at the cell level, DOX@Td@Lipo successfully reversed the resistance of MCF-7/ADR cells to DOX and improved the ability of DOX to kill the tumor cells. The nuclear targeting ability of the constructed system was verified by fluorescence localization. The results showed that, compared with the DOX group, the Td-FAM group, the DOX@Td group and the DOX@Td@Lipo group observed the green fluorescence of FAM and the red fluorescence of DOX in the nucleus of MCF-7/ADR, while DOX and DOX@Lipo groups had no red fluorescence in the nucleus of the nucleus, indicating DOX. DOX@Lipo can not transport DOX into the nucleus of drug-resistant cells, but the DOX@Td@Lipo delivery system can transfer DOX into drug resistant cells and the release of DOX can enter the anti-tumor activity evaluation of the.3.DOX@Td@Lipo cascade drug delivery system of drug resistant cell nuclei: MCF-7/ADR bearing tumor Nude mice were used to evaluate the antitumor activity of the drug delivery system in vivo. The distribution behavior of the delivery system in nude mice was investigated by near infrared imaging technique. The results showed that DOX@Td@Lipo could be rich in tumor tissues after 1 h after intravenous injection, and the drug was enriched in tumor tissue with time. In addition, the results also showed that the encapsulation of immunliposome enhanced the circulation time of the drug in nude mice. The pharmacodynamic results showed that after 2 weeks of treatment, the inhibitory rate of the DOX@Td@Lipo group was 66.7%, the inhibition rate in the group DOX@Lipo was 41.67%, and the group Td@Lipo was not treated. The tumor has a significant therapeutic effect. The antitumor activity of the body is further confirmed by pathology. By comparing the normal tissues and organs, DOX@Td@Lipo significantly reduces the toxic and side effects of DOX.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

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