具有pH響應及細胞核靶向功能的聚合物膠束在癌癥治療中的研究
發(fā)布時間：2018-12-10 06:18
【摘要】：近年來,利用納米載體對癌癥進行靶向治療,在抗癌治療研究中受到了廣泛的關注。為了提高化療藥物的抗癌效果以及減少其對系統(tǒng)的毒副作用,增加腫瘤細胞對納米顆粒的攝取和實現(xiàn)細胞核藥物傳遞顯得尤為重要。考慮利用細胞穿透肽(CPP)修飾具有刺激響應性的納米載體,不僅能達到腫瘤靶向的目的,還能促進腫瘤細胞大量攝取納米顆粒,從而有效抑制癌細胞生長繁殖。在本論文中第二章,我們成功合成了具有細胞穿透肽兼細胞核定位信號Tat修飾的pH響應性聚合物DA-Tat-PEG-PCL(DA-Tat-PECL)。為了改善廣譜抗癌藥物10-羥基喜樹堿(HCPT)的疏水性,提高載藥量(LC)和載藥效率(EE),我們把HCPT接枝在甲氧基聚乙二醇(mPEG)上,成功合成了改性藥物mPEG-HCPT。我們通過核磁共振氫譜(1H NMR),凝膠滲透色譜(GPC),紅外光譜(FT-IR)和高效液相色譜(HPLC)驗證了共聚物結構的正確性。通過溶劑揮發(fā)法,我們制備出具有細胞核靶向和pH響應性的膠束PECL/DA-Tat-M,該膠束具有較低的臨界膠束濃度(CMC),熱力學穩(wěn)定性良好,且具備核殼結構,能有效包載疏水性藥物。納米激光粒度儀DLS和透射電鏡TEM的結果顯示,該膠束呈均一球形,粒徑在80nm左右。體外模擬藥物釋放實驗結果表明:包載mPEG-HCPT的膠束的釋藥速率比包載HCPT的膠束的釋藥速率快,且膠束的LC和EE分別在5%和70%以上。膠束的pH響應性考察結果顯示,在pH 6.8條件下,二甲基馬來酸酐(DA)與Tat之間的酰胺鍵迅速斷裂,Tat完全暴露,使膠束發(fā)生電荷反轉,表面電荷由負變正,且粒徑也增大到90 nm左右。在第三章中,對該電荷反轉膠束PECL/DA-Tat-M進行了一系列體外生物學評價,結果表明PECL/DA-Tat-blank M空白膠束具有良好的生物相容性,載入mPEG-HCPT后能抑制腫瘤細胞,且在pH6.8時具有最低的IC50值。我們發(fā)現(xiàn),腫瘤細胞對該膠束的的攝取,以及亞細胞定位均受到pH的影響,在pH 6.8時,該膠束被腫瘤細胞大量攝取,進入細胞后能有效富集在細胞核周圍,藥物釋放后順利進入細胞核,這些結果均表明,在弱酸性環(huán)境下,DA從共聚物上脫落,Tat暴露出來,促進了細胞的內(nèi)吞作用,進入細胞后,Tat發(fā)揮細胞核定位的作用,使膠束實現(xiàn)細胞核靶向功能。在第四章,建立了 4T1小鼠乳腺癌原位模型和異位模型,用于驗證該膠束的體內(nèi)抗腫瘤效果的研究。結果顯示與對照組,空白膠束組,HCPT組,mPEG-HCPT組合PECL-M膠束組相比,PECL/DA-Tat-M膠束不僅能顯著抑制腫瘤生長,且能集中分布于腫瘤組織,避免全身性的副作用,提高了實驗動物的生存率,有較高的安全性。腫瘤組織的TUNEL染色結果表明,該膠束組的腫瘤細胞凋亡率最高,有最佳抑瘤效果。對原位模型自發(fā)肺轉移的研究結果也表明,PECL/DA-Tat-M膠束抑制了肺轉移,肺部的HE染色分析顯示該組小鼠肺泡結構正常,未發(fā)生肺轉移現(xiàn)象,進一步證明了這種電荷反轉膠束有效的抑制了自發(fā)的肺轉移。
[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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.5
[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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.5
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