【摘要】：惡性腫瘤嚴(yán)重威脅人類生命健康,目前為止,化療仍是治療惡性腫瘤的主要手段,但傳統(tǒng)的化療藥物易產(chǎn)生耐藥,成為化療的主要問題之一。此外,化療藥物還存在腫瘤靶向性差、毒副作用大等缺點。因此,構(gòu)建能夠克服惡性腫瘤耐藥并提高腫瘤靶向性、降低化療藥物毒性的一體化藥物遞送系統(tǒng)(Drug Delivery Systems,DDSs)迫在眉睫。DNA納米技術(shù),作為納米材料科學(xué)的一種新技術(shù),自問世以來,由于其可設(shè)計性和可預(yù)測性,且可形成獨特的形貌特征而備受關(guān)注。其中,DNA四面體(DNA Tetrahedron),具有結(jié)構(gòu)與性能穩(wěn)定、易于多樣化修飾等優(yōu)點,被認(rèn)為是一種理想的藥物載體。脂質(zhì)體(Liposome)具有納米尺度、良好的生物相容性及高度的生物安全性等特征,近年來作為化療藥物的轉(zhuǎn)運載體被廣泛應(yīng)用于靶向藥物遞送領(lǐng)域。細(xì)胞核為多數(shù)化療藥物的主要靶點,但腫瘤細(xì)胞對化療藥物的外排作用導(dǎo)致多數(shù)化療藥物難以到達(dá)細(xì)胞核發(fā)揮作用。細(xì)胞核核孔直徑約為9 nm,而自組裝的DNA四面體粒徑約為5 nm,能夠直接通過核孔高效地進(jìn)入細(xì)胞核中,基于此,本研究首先將臨床一線化療藥物阿霉素(Doxorubicin,DOX)鑲嵌入DNA四面體(Td)中形成DOX@Td;然后,使用免疫脂質(zhì)體將DOX@Td高效包裹,構(gòu)建基于DNA四面體的脂質(zhì)體(DOX@Td@Lipo)高效核轉(zhuǎn)運納米藥物遞送系統(tǒng)。本研究所構(gòu)建的藥物遞送系統(tǒng)的特征為:一、利用免疫脂質(zhì)體將DOX@DNA四面體偶聯(lián)體高效地轉(zhuǎn)運至腫瘤細(xì)胞內(nèi);二、利用DNA四面體小尺寸(小于細(xì)胞核孔)及核內(nèi)可降解特性,將DOX高效的轉(zhuǎn)運至細(xì)胞核中,有效地避免了腫瘤細(xì)胞的藥物外排途徑,逆轉(zhuǎn)腫瘤細(xì)胞耐藥。該系統(tǒng)顯著地提高了DOX對耐藥乳腺癌的治療效率,并降低了DOX的毒副作用。然后,本研究以人類乳腺癌細(xì)胞MCF-7和人類耐藥乳腺癌細(xì)胞MCF-7/ADR為細(xì)胞模型以及以MCF-7/ADR荷瘤BLAB/C裸鼠為動物模型,主要進(jìn)行了以下三方面的研究:1.DOX@Td@Lipo級聯(lián)式藥物遞送系統(tǒng)的制備及表征:單鏈DNA(ssDNA)復(fù)性并堿基互補配對自組裝成DNA四面體,原子力顯微鏡(Automatic Force Microscope,AFM)以及凝膠成像表征驗證,結(jié)果顯示制備四面體粒徑在5 nm左右,且分布均勻;利用DOX易于插入到DNA雙螺旋結(jié)構(gòu)的性質(zhì),制備DOX@Td偶聯(lián)物,通過電泳實驗考察其穩(wěn)定性,結(jié)果表明偶聯(lián)DOX后的DNA四面體仍維持其四面體結(jié)構(gòu),具有較強(qiáng)穩(wěn)定性;最終,采用逆向蒸發(fā)法制備DOX@Td@Lipo,用透射電子顯微鏡(Transmission Electron Microscope,TEM)對其表征,粒徑在147 nm左右,并且粒徑分布均勻。2.DOX@Td@Lipo級聯(lián)式藥物遞送系統(tǒng)體外抗腫瘤活性及逆轉(zhuǎn)腫瘤細(xì)胞耐藥研究:系統(tǒng)考察DOX@Td@Lipo對MCF-7及MCF-7/ADR細(xì)胞的抗腫瘤活性,結(jié)果表明對于MCF-7細(xì)胞,DOX、DOX@Lipo以及DOX@Td@Lipo對MCF-7細(xì)胞的增殖抑制作用無顯著性差別(48 h時抑制率分別為70.65%、69.55%、65.08%)。然而,DOX和DOX@Lipo組對ADR細(xì)胞的增殖抑制作用顯著低于DOX@Td@Lipo組(48h時抑制率分別為21.23%、25.73%、79.83%)。表明在細(xì)胞水平,DOX@Td@Lipo成功地逆轉(zhuǎn)了MCF-7/ADR細(xì)胞對DOX的耐藥性進(jìn)而提高了DOX殺傷腫瘤細(xì)胞的能力。此外,本研究還通過熒光定位的方法對所構(gòu)建系統(tǒng)的細(xì)胞核靶向能力進(jìn)行驗證。結(jié)果顯示,與DOX組相比,Td-FAM組、DOX@Td組及DOX@Td@Lipo組在MCF-7/ADR細(xì)胞核部位觀測到FAM的綠色熒光和DOX的紅色熒光,而DOX組和DOX@Lipo組在細(xì)胞核部位沒有紅色熒光出現(xiàn)。說明DOX不能進(jìn)入耐藥細(xì)胞細(xì)胞核,DOX@Lipo也不能將DOX運送入耐藥細(xì)胞的細(xì)胞核,但是DOX@Td@Lipo藥物遞送系統(tǒng)能夠?qū)OX轉(zhuǎn)運入耐藥細(xì)胞并且釋放的DOX能夠進(jìn)入耐藥細(xì)胞細(xì)胞核。3.DOX@Td@Lipo級聯(lián)式藥物遞送系統(tǒng)體內(nèi)抗腫瘤活性評價:以MCF-7/ADR荷瘤裸鼠為模型,評價所構(gòu)建的藥物遞送系統(tǒng)的體內(nèi)抗腫瘤活性。通過近紅外光活體成像技術(shù)考察所構(gòu)建的遞送系統(tǒng)在荷瘤裸鼠體內(nèi)的分布行為,結(jié)果顯示,靜脈注射后1 h后DOX@Td@Lipo即可在腫瘤組織富集,且隨時間延長富集在腫瘤組織的藥物越多。此外,結(jié)果還表明,免疫脂質(zhì)體的包封增強(qiáng)了藥物在裸鼠體內(nèi)的循環(huán)時間。藥效學(xué)結(jié)果顯示,經(jīng)過2周治療,制劑DOX@Td@Lipo組對腫瘤的抑制率為66.7%,DOX@Lipo組抑制率41.67%,Td@Lipo組無治療作用。說明所構(gòu)建的藥物遞送系統(tǒng)對乳腺癌耐藥腫瘤具有顯著的治療作用。體內(nèi)抗腫瘤活性也通過病理學(xué)進(jìn)行了進(jìn)一步確證,通過對比正常組織器官發(fā)現(xiàn),DOX@Td@Lipo顯著降低了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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943;R96

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