腫瘤微環(huán)境響應性納米膠束的制備及其用于“抗癌前藥—酶”的聯合輸送研究
發(fā)布時間：2018-07-25 21:09
【摘要】：癌癥已經成為世界人口死亡的主要原因之一。目前,癌癥的主要治療方法有化學療法、光熱療法和輻射療法等等,已經得到了廣泛的研究。近年來,“酶-前藥”療法(“enzyme-prodrug”therapy,EPT)作為一種新的癌癥治療策略,已展現出很好的癌癥治療效果,并得到了大量的臨床驗證。EPT是一種兩步療法:首先,使用靶向策略將前藥活化酶傳輸到腫瘤部位;其次,將無毒的前藥靜脈注射后通過血液循環(huán)傳遞到腫瘤組織,無毒的抗腫瘤前藥就會在前藥活化酶的催化下產生活性抗腫瘤藥物。雖然“酶-抗腫瘤前藥”療法為人們展示了廣闊的應用前景,給腫瘤治療帶來了一個新思路,但仍有許多關鍵性問題亟待解決:“酶-前藥”療法需要兩步治療,這就增加了病人的痛苦與治療成本,并且第二次用藥的時間也不易把握(對于抗體導向“酶-前藥”療法,首先將抗體-酶偶聯物靜脈注入體內,借助抗體識別腫瘤表面抗原的特性,使酶被帶到腫瘤靶位,然后當抗體-酶偶聯物從血液中清除后才能靜脈注入無抗癌活性或低活性的前藥,不然滯留在體內正常組織器官的酶就會催化前藥,進而對正常組織造成損傷,因此對于第二次用藥的時間就不易把握)�！懊�-前藥”療法中前藥的用藥劑量比較大,這是由于藥物在病變部位只有達到一定濃度才能發(fā)揮治療作用,而藥物在體內傳輸過程中會有滯留等現象,給藥劑量太小就會造成到達病變組織藥物濃度不夠,但用藥劑量太大則會造成藥物的蓄積,可能對機體造成一定的毒副作用,并且增加了治療成本。如果酶和前藥被同時地輸送到腫瘤細胞,由兩步療法所造成的缺陷就會被極大地避免。所以,發(fā)展一種有效的能夠同時傳輸酶和前藥的載體是極其重要的。辣根過氧化物酶-吲哚乙酸(IAA-HRP)體系是近年來研究比較多的一種“酶-前藥”系統。IAA作為一種植物激素,被HRP氧化后會產生包括活性氧自由基(ROS),如吲哚基、3-甲基吲哚基以及過氧化基在內的自由基。其可以通過引起細胞脂質過氧化引起細胞膜結構改變、DNA損傷、降低Bcl-2活性以及激活凋亡相關基因,導致細胞內損傷和凋亡。然而,相同濃度IAA對正常細胞生長的影響并不顯著,IAA也不易被哺乳動物的過氧化物酶氧化。酯鍵是前藥設計中一種非常普遍的鍵接方式,其可在人體酯酶的作用下發(fā)生水解。吲哚乙酸乙酯(EIA)是一種IAA的衍生物,當其被傳輸到腫瘤組織后,在酯酶及腫瘤部位低的pH作用下發(fā)生水解,產生前藥IAA。由兩親性大分子通過自組裝形成的聚合物膠束,在傳遞抗腫瘤藥物、siRNA以及其它治療劑方面有很大的潛力。納米膠束可以通過EPR效應優(yōu)先進入到腫瘤組織,大大減少了化療的副作用,同時,膠束有較長的血液循環(huán)時間,可提高藥物的利用率。此外,聚合物膠束有對于腫瘤微環(huán)境的可設計性。例如,腫瘤組織(pH 6.5-6.8)、核內體(pH 5.5-6.5)和溶酶體(pH 4.5-5.5)是微酸性性環(huán)境,不同于血液和正常組織(pH 7.4)。因此,pH敏感型膠束可以實現腫瘤部位藥物的智能可控釋放。此外,腫瘤部位細胞內還原性谷胱甘肽(GSH)的濃度是細胞外GSH濃度的100-1000倍。含二硫鍵鏈的還原敏感型聚合物膠束在腫瘤部位會發(fā)生解體,膠束載體被迅速破壞,可實現藥物在腫瘤部位可控釋放。因此,聚合物膠束可以作為理想的載體來實現酶和藥物的聯合輸送。本論文主要通過設計不同腫瘤微環(huán)境敏感型的聚合物膠束,來實現酶和前藥的聯合輸送,從而避免了兩步“酶-前藥”療法的缺陷。具體內容包括:(1)設計合成了還原敏感型三嵌段共聚物mPEG-PAsp(AED)-CA,自組裝形成納米膠束,共負載了HRP和EIA。PEG為膠束的外殼層,HRP通過靜電作用于PAsp(AED)段的氨基上,EIA通過疏水作用包覆于膠束核層內。當膠束到達腫瘤部位后,聚合物膠束PAsp(AED)段上的二硫鍵在還原型谷胱甘肽的作用下斷裂,膠束結構被破壞,HRP和EIA得到釋放。EIA水解產生IAA,IAA被HRP激活將產生大量的ROS,殺死腫瘤細胞。通過透射電鏡(TEM)測得負載HRP和EIA的膠束尺寸大約為40 nm,細胞存活率測試結果表明HRP/EIA膠束對人體肺腺癌細胞有很強的殺傷力,證明一步“酶-前藥”療法具有很大的潛力。(2)設計合成了pH敏感型三嵌段共聚物mPEG-PAsp(THA)-AcMal7,自組裝形成納米膠束,共負載了HRP和EIA。PEG為膠束外殼,PAsp(THA)段可靜電吸附HRP,形成膠束次外層,AcMal7為pH敏感型疏水鏈段,用于形成疏水型的膠束內核來包覆EIA。當聚合物膠束到達腫瘤部位后,在腫瘤低的pH作用下,AcMal7脫保護由疏水變?yōu)橛H水,此時,膠束結構被破壞,EIA和HRP得到釋放,進而產生大量ROS,殺死腫瘤細胞。通過TEM對膠束形態(tài)進行了表征,通過MTT對細胞活率進行了測試,結果表明所制備pH敏感型聚合物膠束在酶前藥一步療法中具有很大的優(yōu)勢,與上一個工作相比,避免了氨基還原性對自由基的消耗,提高了治療效率。
[Abstract]:Cancer has become one of the major causes of death in the world. At present, the main treatments for cancer are chemotherapy, photothermal therapy and radiation therapy, and so on. It has been widely studied. In recent years, "enzyme-prodrug" ("enzyme" therapy, EPT) as a new cancer treatment strategy, has shown good cancer. .EPT is a two step therapy: first, using the target strategy to transfer the prodrug activator to the tumor site; secondly, the nontoxic prodrug is injected through the blood circulation to the tumor tissue, and the nontoxic antitumor prodrug will produce a living anti swelling under the catalysis of the precursor activator. Tumor drugs. Although the "enzyme antiantineoantineoantineodrug" therapy has shown a broad prospect of application and brings a new idea to the treatment of cancer, there are still many key problems to be solved: "enzyme prodrug" therapy requires two steps of treatment, which increases the patient's pain and treatment cost, and the time of the second medication is not easy to take. For the antibody directed enzyme - prodrug therapy, the antibody enzyme coupling is injected into the body and the antibody identification of the tumor surface antigen is used to identify the tumor surface antigen, so that the enzyme is taken to the tumor target and then the antibody enzyme coupling is removed from the blood until the anticancer or low activity prodrug is injected into the body, otherwise the body is detained in the body. The enzymes that organize the organs will catalyze the prodrugs and then cause damage to the normal tissue, so the time for the second drug use is not easy to grasp. The dosage of the drug in the "enzyme pre drug" therapy is relatively large, because the drug can be used only to reach a certain concentration in the lesion, and the drug will be transmitted in the body. If the dosage is too small, the drug concentration is too small to cause the drug concentration to reach the lesion, but the dosage is too large will cause the accumulation of drugs, may cause certain toxic side effects to the body, and increase the cost of treatment. If the enzyme and the prodrug are transported to the tumor cells at the same time, the defects caused by the two step therapy will be Therefore, it is very important to develop an effective carrier that can transmit enzymes and prodrugs at the same time. The horseradish peroxidase indoloacetic acid (IAA-HRP) system is a kind of "enzyme - prodrug" system.IAA as a plant hormone, which is oxidized by HRP to include reactive oxygen free radicals (ROS), such as The free radicals, such as indolyl, 3- methyl indolyl and peroxidation, can cause cell membrane structure change, DNA damage, Bcl-2 activity and activation of apoptosis related genes by causing lipid peroxidation in cells, causing intracellular damage and apoptosis. However, the effect of the same concentration of IAA on normal cell growth is not significant, and IAA does not Peroxidase is easily oxidized by mammalian peroxidase. Ester bond is a very common bonding method in the design of prodrug. It can be hydrolyzed under the action of human esterase. EIA is a derivative of IAA. When it is transmitted to the tumor tissue, it is hydrolyzed under the action of esterase and the low pH of the tumor, producing the prodrug IAA. The polymer micelles formed by two Pro macromolecules through self-assembly have great potential in the delivery of antitumor drugs, siRNA and other therapeutic agents. Nanomicelles can first enter the tumor tissue through the EPR effect, greatly reducing the side effects of chemotherapy, while the micelle has a long blood circulation time, which can improve the benefit of the drug. In addition, polymer micelles have a design for tumor microenvironment. For example, the tumor tissue (pH 6.5-6.8), the pH 5.5-6.5 (pH 5.5-6.5) and the lysosome (pH 4.5-5.5) are microacid environments, different from the blood and normal tissues (pH 7.4). Therefore, the pH sensitive adhesive bundles can realize the intelligent controlled release of the tumor site drugs. In addition, the tumor part The concentration of reduced glutathione (GSH) in the cell is 100-1000 times that of the extracellular GSH. The reduction sensitive polymer micelles containing two sulfur bonds are disintegrated at the tumor site, and the micellar carrier is rapidly destroyed and the drug can be controlled to release in the tumor site. Therefore, the polymer micelle can be used as an ideal carrier to realize the enzyme and the enzyme. Combined transport of drugs. In this paper, the polymer micelles of different tumor microenvironment sensitive types were designed to combine the enzyme with the precursor, thus avoiding the defects of the two step "enzyme prodrug" therapy. The specific contents include: (1) the design and synthesis of the reduction sensitive three block copolymer mPEG-PAsp (AED) -CA and the self-assembled formation of nanoscale Micelles, CO loaded with HRP and EIA.PEG as the shell layer of the micelle, HRP by electrostatic action on the amino group of the PAsp (AED) section, EIA is coated in the nuclear layer of the micelle by hydrophobicity. When the micelle reaches the tumor site, the two sulfur bond on the PAsp (AED) segment of the polymer micelle breaks under the effect of the cystamine in the prototype Valley, the micelle structure is destroyed, HRP and EIA obtained. By releasing.EIA hydrolysis to produce IAA and IAA being activated by HRP, a large number of ROS will be produced to kill tumor cells. The micellar size of the loaded HRP and EIA is estimated to be about 40 nm by transmission electron microscopy (TEM). The cell viability test results show that the HRP/EIA micelle has a strong killing force on human lung adenocarcinoma cells, proving that one step "enzyme prodrug" therapy is very large. (2) (2) the design and synthesis of pH sensitive three block copolymer mPEG-PAsp (THA) -AcMal7, self-assembled to form nano micelle, CO loaded with HRP and EIA.PEG as micellar shell, PAsp (THA) segment can be electrostatic adsorbed HRP, form a micellar secondary layer, AcMal7 for pH sensitive hydrophobic chain section, to form a hydrophobic micellar core to encapsulate EIA. when polymer glue After the beam reaches the tumor site, the AcMal7 deprotection is changed from hydrophobicity to hydrophilic under the low pH action of the tumor. At this time, the micelle structure is destroyed, EIA and HRP are released, and a large number of ROS are produced and the tumor cells are killed. The micelle morphology is characterized by TEM and the cell viability is tested by MTT. The results show that the pH sensitive polymerization is prepared. The micelle has a great advantage in the one step therapy of enzyme predrug. Compared with the previous work, it avoids the consumption of amino reducibility to free radicals and improves the efficiency of treatment.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943
[Abstract]:Cancer has become one of the major causes of death in the world. At present, the main treatments for cancer are chemotherapy, photothermal therapy and radiation therapy, and so on. It has been widely studied. In recent years, "enzyme-prodrug" ("enzyme" therapy, EPT) as a new cancer treatment strategy, has shown good cancer. .EPT is a two step therapy: first, using the target strategy to transfer the prodrug activator to the tumor site; secondly, the nontoxic prodrug is injected through the blood circulation to the tumor tissue, and the nontoxic antitumor prodrug will produce a living anti swelling under the catalysis of the precursor activator. Tumor drugs. Although the "enzyme antiantineoantineoantineodrug" therapy has shown a broad prospect of application and brings a new idea to the treatment of cancer, there are still many key problems to be solved: "enzyme prodrug" therapy requires two steps of treatment, which increases the patient's pain and treatment cost, and the time of the second medication is not easy to take. For the antibody directed enzyme - prodrug therapy, the antibody enzyme coupling is injected into the body and the antibody identification of the tumor surface antigen is used to identify the tumor surface antigen, so that the enzyme is taken to the tumor target and then the antibody enzyme coupling is removed from the blood until the anticancer or low activity prodrug is injected into the body, otherwise the body is detained in the body. The enzymes that organize the organs will catalyze the prodrugs and then cause damage to the normal tissue, so the time for the second drug use is not easy to grasp. The dosage of the drug in the "enzyme pre drug" therapy is relatively large, because the drug can be used only to reach a certain concentration in the lesion, and the drug will be transmitted in the body. If the dosage is too small, the drug concentration is too small to cause the drug concentration to reach the lesion, but the dosage is too large will cause the accumulation of drugs, may cause certain toxic side effects to the body, and increase the cost of treatment. If the enzyme and the prodrug are transported to the tumor cells at the same time, the defects caused by the two step therapy will be Therefore, it is very important to develop an effective carrier that can transmit enzymes and prodrugs at the same time. The horseradish peroxidase indoloacetic acid (IAA-HRP) system is a kind of "enzyme - prodrug" system.IAA as a plant hormone, which is oxidized by HRP to include reactive oxygen free radicals (ROS), such as The free radicals, such as indolyl, 3- methyl indolyl and peroxidation, can cause cell membrane structure change, DNA damage, Bcl-2 activity and activation of apoptosis related genes by causing lipid peroxidation in cells, causing intracellular damage and apoptosis. However, the effect of the same concentration of IAA on normal cell growth is not significant, and IAA does not Peroxidase is easily oxidized by mammalian peroxidase. Ester bond is a very common bonding method in the design of prodrug. It can be hydrolyzed under the action of human esterase. EIA is a derivative of IAA. When it is transmitted to the tumor tissue, it is hydrolyzed under the action of esterase and the low pH of the tumor, producing the prodrug IAA. The polymer micelles formed by two Pro macromolecules through self-assembly have great potential in the delivery of antitumor drugs, siRNA and other therapeutic agents. Nanomicelles can first enter the tumor tissue through the EPR effect, greatly reducing the side effects of chemotherapy, while the micelle has a long blood circulation time, which can improve the benefit of the drug. In addition, polymer micelles have a design for tumor microenvironment. For example, the tumor tissue (pH 6.5-6.8), the pH 5.5-6.5 (pH 5.5-6.5) and the lysosome (pH 4.5-5.5) are microacid environments, different from the blood and normal tissues (pH 7.4). Therefore, the pH sensitive adhesive bundles can realize the intelligent controlled release of the tumor site drugs. In addition, the tumor part The concentration of reduced glutathione (GSH) in the cell is 100-1000 times that of the extracellular GSH. The reduction sensitive polymer micelles containing two sulfur bonds are disintegrated at the tumor site, and the micellar carrier is rapidly destroyed and the drug can be controlled to release in the tumor site. Therefore, the polymer micelle can be used as an ideal carrier to realize the enzyme and the enzyme. Combined transport of drugs. In this paper, the polymer micelles of different tumor microenvironment sensitive types were designed to combine the enzyme with the precursor, thus avoiding the defects of the two step "enzyme prodrug" therapy. The specific contents include: (1) the design and synthesis of the reduction sensitive three block copolymer mPEG-PAsp (AED) -CA and the self-assembled formation of nanoscale Micelles, CO loaded with HRP and EIA.PEG as the shell layer of the micelle, HRP by electrostatic action on the amino group of the PAsp (AED) section, EIA is coated in the nuclear layer of the micelle by hydrophobicity. When the micelle reaches the tumor site, the two sulfur bond on the PAsp (AED) segment of the polymer micelle breaks under the effect of the cystamine in the prototype Valley, the micelle structure is destroyed, HRP and EIA obtained. By releasing.EIA hydrolysis to produce IAA and IAA being activated by HRP, a large number of ROS will be produced to kill tumor cells. The micellar size of the loaded HRP and EIA is estimated to be about 40 nm by transmission electron microscopy (TEM). The cell viability test results show that the HRP/EIA micelle has a strong killing force on human lung adenocarcinoma cells, proving that one step "enzyme prodrug" therapy is very large. (2) (2) the design and synthesis of pH sensitive three block copolymer mPEG-PAsp (THA) -AcMal7, self-assembled to form nano micelle, CO loaded with HRP and EIA.PEG as micellar shell, PAsp (THA) segment can be electrostatic adsorbed HRP, form a micellar secondary layer, AcMal7 for pH sensitive hydrophobic chain section, to form a hydrophobic micellar core to encapsulate EIA. when polymer glue After the beam reaches the tumor site, the AcMal7 deprotection is changed from hydrophobicity to hydrophilic under the low pH action of the tumor. At this time, the micelle structure is destroyed, EIA and HRP are released, and a large number of ROS are produced and the tumor cells are killed. The micelle morphology is characterized by TEM and the cell viability is tested by MTT. The results show that the pH sensitive polymerization is prepared. The micelle has a great advantage in the one step therapy of enzyme predrug. Compared with the previous work, it avoids the consumption of amino reducibility to free radicals and improves the efficiency of treatment.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943
【相似文獻】
相關期刊論文 前10條
1 何慶,盛京;響應性凝膠及其在藥物控釋上的應用[J];功能高分子學報;1997年01期
2 王濤;胡小波;劉新星;童真;;納米復合水凝膠的力學行為與刺激響應性[J];中國科學:化學;2012年05期
3 呂少一;邵自強;張振玲;王飛俊;王文俊;;新型刺激響應性纖維素基含能凝膠的流變性能[J];高等學�；瘜W學報;2012年02期
4 陳朝霞;張玉紅;劉航;艾書倫;何培新;;環(huán)境響應性微球研究進展[J];粘接;2013年01期
5 馬永翠;唐剛;閔曉燕;李杰;楊耀宗;畢韻梅;;溫度響應性樹枝化聚合物研究進展[J];化學世界;2013年04期
6 范會強,廖列文,涂星,管海鳳;刺激響應性水凝膠的研究現狀及發(fā)展趨勢[J];上�；�;2003年06期
7 胡亞薇;王小濤;李振華;龔興厚;葉曉鐵;杜飛鵬;劉最芳;;基于控制釋放的光響應性聚合物微納米粒子的研究進展[J];功能材料;2014年17期
8 閆強;趙越;;二氧化碳響應性聚合物及其應用進展[J];高分子材料科學與工程;2014年02期
9 湯飛;鐘凱;鄭新威;;刺激響應性磁共振成像造影劑的研究進展[J];化工新型材料;2014年06期
10 周希蕊;陳龍;羅施中;;環(huán)境響應性多肽自組裝研究進展[J];中國科學:化學;2013年08期
相關會議論文 前10條
1 王彥彥;劉利彬;喬從德;李天鐸;;N-取代不同烷基聚氨酯膜的刺激響應性研究[A];中國化學會第十四屆膠體與界面化學會議論文摘要集-第1分會：表面界面與納米結構材料[C];2013年
2 葛瑞;羅施中;;環(huán)境響應性高分子-多肽接合物材料的制備、表征及應用[A];第八屆全國化學生物學學術會議論文摘要集[C];2013年
3 劉世勇;;響應性聚合物組裝體的構筑與功能拓展[A];2013年全國高分子學術論文報告會論文摘要集——大會邀請報告[C];2013年
4 李闖;楊忠強;劉冬生;;多重響應性、力學強度可調的Polypeptide-DNA水凝膠[A];2013年全國高分子學術論文報告會論文摘要集——主題I：生物高分子與天然高分子[C];2013年
5 王力彥;趙趲;殷亮;袁港;;兩個溫度響應性均聚物的層層組裝和多層膜的低溫溶解性[A];中國化學會第28屆學術年會第15分會場摘要集[C];2012年
6 黃娟;薛亞楠;張寒;溫凱凱;羅曉剛;蔡寧;喻發(fā)全;;還原響應性海藻酸鈉/四氧化三鐵載藥納米凝膠的制備與表征[A];2013年全國高分子學術論文報告會論文摘要集——主題F：功能高分子[C];2013年
7 余雷曉;關悅;劉洋;陳思，
本文編號：2145095
本文鏈接：http://www.sikaile.net/yixuelunwen/yiyaoxuelunwen/2145095.html
最近更新
教材專著
