pH敏感聚（原酸酯—氨酯）药物载体的合成、表征及体内外抗肿瘤评估
发布时间：2018-07-29 05:50
【摘要】：生物降解性聚合物纳米药物载体因具有较高的载药率和包封率,良好的体内药物分布及降低药物毒副作用的能力,延长药物的体内循环时间等优点而被广泛应用于抗肿瘤药物传递。如今,研究者们已经开发了一系列高分子材料用于抗肿瘤药物传递。其中生物降解性聚氨酯作为一种人工合成的生物相容性高分子材料,因其制备方法简单、高效和优越的生物医学性能而被广泛应用于各种生物医学领域,主要包括药物缓控系统和组织工程支架。传统的聚氨酯通过引入降解性聚酯或聚碳酸酯而被赋予降解性,然而这些聚氨酯的降解速率较慢,往往不能满足抗肿瘤药物传递的要求。为了提高传统聚氨酯的降解速率,研究者们将一些环境响应性的化学键(例如:pH敏感缩酮键、缩醛键、腙键和还原敏感的二硫键等)引入到了聚氨酯的主链中。原酸酯键作为一种微酸敏感的化学键,已被广泛应用到抗肿瘤纳米药物载体中。然而主链含原酸酯的pH敏感聚氨酯的制备及其在抗肿瘤纳米药物载体领域的应用还未见报道。本论文通过一种酸敏感的原酸酯二氨基单体和两种分子量不同的聚己内酯二醇(Fw=530和2000)活性酯在温和的条件下缩聚得到2种pH敏感聚(原酸酯-氨酯)(POEU1和POEU2),并通过水包油(O/W)单层乳液技术分别制备了具有不同酸降解速率的纳米粒子(NP1和NP2)。通过核磁以及凝胶渗透色谱(GPC)检测聚(原酸酯-氨酯)的结构以及分子量,结果表明POEU1和POEU2结构正确,数均分子量(Mn)分别为2.2×104和4.09104。透射电镜(TEM)以及动态光衍射仪(DLS)结果表明,两种纳米粒子粒径分布在200 nm左右且都为球形结构。体外降解结果表明两种纳米粒子在生理条件(pH 7.4)下非常稳定,而在弱酸性条件下(pH 5.0)持续降解并伴随着粒径的不断减小。另外,在pH 5.0条件下,两种纳米粒子(NP1和NP2)的降解速率可被聚合物的疏水性调控。在192 h后,由疏水性较弱的POEU1制备的纳米粒子(NP1)完全降解,而由疏水性较强的POEU2制备的纳米粒子(NP2)仅降解了 83.41%。由POEU1和POEU2制备的纳米粒子(NP1和NP2)可高效的包载抗肿瘤药物阿霉素(DOX),得到了两种载药纳米粒子,分别命名为NP1/DOX和NP2/DOX。体外药物释放实验结果表明:NP1/DOX和NP2/DOX具有显著地酸加速药物释放行为,并且释放速率可以被聚合物的疏水性调控。在pH 5.0,由疏水性强的POEU2制备的NP2/DOX释放速率慢于由疏水性相对弱的POEU1制备的NP1/DOX。通过激光共聚焦扫描显微镜(CLSM)以及流式细胞仪分别定性和定量检测了人神经母细胞瘤细胞(SH-SY5Y)和鼠源肝癌细胞(H22)对NP1/DOX和NP2/DOX的摄取情况,结果表明NP1/DOX和NP2/DOX可被SH-SY5Y和H22细胞有效地摄取并释放出阿霉素进入细胞核。通过MTT法检测空白纳米粒子NP1和NP2以及载药粒子NP1/DOX和NP2/DOX与SH-SY5Y和H22细胞共培养24 h后的细胞存活率。结果显示,对于空白纳米粒子,即使在最大浓度1mg/mL,细胞的存活率都在90%以上,表明空白纳米粒子NP1和NP2基本无毒。然而,NP1/DOX和NP2/DOX可以有效地杀死癌细胞SH-SY5Y和H22,并呈现出浓度依赖性杀伤作用,即随着载药粒子浓度升高,SH-SY5Y和H22细胞存活率降低。由人神经母细胞瘤细胞形成的3维多细胞球(3D SH-SY5Y MCTS)作为一种体外肿瘤模型被用于模拟NP1/DOX和NP2/DOX对肿瘤的渗透和生长抑制作用。结果表明,随着NP1/DOX和NP2/DOX与SH-SY5Y MCTS共培养时间的延长,相比于裸药(DOX),NP1/DOX和NP2/DOX对SH-SY5Y MCTS表现出了更好的渗透以及更高的生长抑制作用。由鼠源肝癌细胞H22建立的肝癌小鼠模型被用来检测两种载药纳米粒子NP1/DOX和NP2/DOX在体内各器官及肿瘤组织的药物分布,毒副作用以及抗肿瘤效果。体内药物分布结果表明,与裸药(DOX)相比,包载在纳米粒子中的阿霉素具有更长的血液循环时间,更有效的肿瘤富集以及更低的心脏毒性。体内抗肿瘤结果表明,在小鼠处理的7天内,生理盐水组以及空白纳米粒子组(NP1和NP2)的小鼠肿瘤不断增大,7天后肿瘤体积增加了 8-9倍,然而裸药(DOX)组以及两种载药粒子组(NP1/DOX和NP2/DOX)的小鼠肿瘤被明显抑制,且NP1/DOX 和 NP2/DOX 抑制率(72.68%和 70.37%)高于裸药(DOX)组(62.61%)。最后利用组织切片技术进一步评估NP1/DOX和NP2/DOX的毒副作用以及抗肿瘤效果,结果显示,相比于生理盐水组,裸药(DOX)组和两种载药粒子(NP1/DOX和NP2/DOX)组小鼠的肿瘤组织细胞数目显著下降并出现了不同程度的坏死,并且这一现象在NP1/DOX和NP2/DOX给药组更为严重。另外裸药(DOX)组的小鼠心脏坏死严重,然而NP1/DOX和NP2/DOX组小鼠的心脏基本正常且也没有其他器官毒性。综上所述,这种生物相容性良好的pH敏感聚(原酸酯-氨酯)在抗肿瘤药物传递领域具有良好的应用前景。
[Abstract]:Biodegradable polymer nanomaterials are widely used in antitumor drug delivery because of their high drug loading rate and encapsulation efficiency, good drug distribution in the body and the ability to reduce drug side effects and prolong the circulation time of drugs. Biodegradable polyurethane, in which biodegradable polyurethane is a synthetic biocompatible polymer material, is widely used in various biomedical fields because of its simple preparation method, high efficiency and superior biomedical properties, including drug slow control system and tissue engineering scaffold. Degrading polyesters or polycarbonate are endowed with degradability, but the degradation rate of these polyurethanes is slow and often fails to meet the requirements for the transfer of antitumor drugs. In order to improve the degradation rate of traditional polyurethane, researchers have some environmental responsive chemical bonds (such as pH sensitive ketal bond, acetal bond, hydrazone bond and reduction sensitive two sulfur). Bonds, etc., are introduced into the main chain of polyurethane. As a kind of acid sensitive chemical bond, the original ester bond has been widely used in anti-tumor nano drug carriers. However, the preparation of pH sensitive polyurethane and its application in the field of anti-tumor nanoscale drug carriers have not yet been reported. The two amino monomers and two kinds of polyhexyl glycol (Fw=530 and 2000) active esters with different molecular weights were polycondensation under mild conditions to obtain 2 pH sensitive poly (POEU1 and POEU2), and nanoparticles (NP1 and NP2) with the rate of inhomogeneous degradation were prepared by the monolayer emulsion of water oil (O/W). The structure and molecular weight of poly (tryptophone) were detected by gel permeation chromatography (GPC). The results showed that the structure of POEU1 and POEU2 was correct. The number of molecular weights (Mn) were 2.2 x 104 and 4.09104. transmission electron microscopy (TEM) and dynamic light diffractometer (DLS). The results showed that the particle size distribution of two nanoparticles was around 200 nm and all were spherical. The results show that the two nanoparticles are very stable under the physiological conditions (pH 7.4), while under the weak acid condition (pH 5), the degradation rate and the particle size decrease continuously. In addition, the degradation rate of the two nanoparticles (NP1 and NP2) can be controlled by the hydrophobicity of the polymer under the condition of pH 5. After 192 h, the weak hydrophobic POEU1 is prepared. The nanoparticles (NP1) were completely degraded, and the nanoparticles (NP2), prepared by the hydrophobic POEU2, only degraded the nanoparticles (NP1 and NP2) prepared by POEU1 and POEU2 (NP1 and NP2), which could efficiently encapsulate the antitumor drug adriamycin (DOX). Two kinds of drug loaded nanoparticles were obtained, named as NP1/DOX and NP2/DOX. drug release experimental nodes, respectively. The results showed that NP1/DOX and NP2/DOX significantly accelerated the drug release behavior, and the release rate could be regulated by the hydrophobicity of the polymer. In pH 5, the release rate of NP2/DOX prepared by the hydrophobic POEU2 was slower than the NP1/DOX. which was prepared by the relatively weak hydrophobic POEU1 through the stimulated confocal scanning microscope (CLSM) and the flow cells. The uptake of NP1/DOX and NP2/DOX by human neuroblastoma cells (SH-SY5Y) and mouse derived hepatoma cells (H22) was detected and quantified. The results showed that NP1/DOX and NP2/DOX could be effectively absorbed and released by SH-SY5Y and H22 cells and released adriamycin into the nucleus. The MTT method was used to detect NP1 and NP2 and drug carrier particles by MTT method. The survival rates of NP1/DOX and NP2/DOX with SH-SY5Y and H22 cells were co cultured for 24 h. The results showed that the survival rate of the cells was more than 90% for the blank nanoparticles, even at the maximum concentration of 1mg/mL, indicating that the blank nanoparticles NP1 and NP2 were basically non-toxic. However, NP1/DOX and NP2/ DOX could effectively kill cancer cell SH-SY5Y and cells. There is a concentration dependent killing effect, that is, the survival rate of SH-SY5Y and H22 cells decreases with the increase of the concentration of drug particles. The 3 dimensional multicellular ball (3D SH-SY5Y MCTS) formed by the human neuroblastoma cells is used as an in vitro tumor model to simulate the infiltration and growth inhibition of NP1/DOX and NP2/DOX to the tumor. The results show that with NP1, with NP1 The co culture time of /DOX and NP2/DOX and SH-SY5Y MCTS was prolonged, compared with the naked drug (DOX), NP1/DOX and NP2/DOX showed better permeability and higher growth inhibition to SH-SY5Y MCTS. The mouse model of liver cancer, established by mouse hepatoma cell H22, was used to detect two kinds of drug loaded nanoparticles NP1/DOX and NP2/DOX in the organs and swelling in the body. The distribution, side effects, and antitumor effects of the tumor tissue. The drug distribution in vivo shows that adriamycin, which is loaded in the nanoparticles, has longer blood circulation time, more effective tumor enrichment and lower cardiac toxicity compared with the naked drug (DOX). In vivo antitumor results show that within 7 days of the mice treated, the physiological saline The mice tumor in the group and the blank nanoparticles group (NP1 and NP2) increased continuously, and the tumor volume increased by 8-9 times in 7 days. However, the tumor in the naked drug (DOX) group and the two carrier group (NP1/DOX and NP2/DOX) were obviously suppressed, and the inhibition rate of NP1/DOX and NP2/DOX (72.68% and 70.37%) was higher than that of the naked drug (62.61%) group (62.61%). Finally, the tissue was used in the tissue. The slicing technique further assessed the side effects and antitumor effects of NP1/DOX and NP2/DOX. The results showed that the number of tumor tissue cells in the naked drug (DOX) group and the two drug loaded particles (NP1/DOX and NP2/DOX) mice decreased significantly and had varying degrees of necrosis compared to the saline group, and this phenomenon was given in NP1/DOX and NP2/DOX. In addition, the mice in the DOX group had serious heart necrosis. However, the hearts of the NP1/DOX and NP2/DOX mice were basically normal and no other organ toxicity. In conclusion, the good biocompatible pH sensitive poly (tryptophone) has a good application prospect in the field of antitumor drug delivery.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5
本文编号：2151726
[Abstract]:Biodegradable polymer nanomaterials are widely used in antitumor drug delivery because of their high drug loading rate and encapsulation efficiency, good drug distribution in the body and the ability to reduce drug side effects and prolong the circulation time of drugs. Biodegradable polyurethane, in which biodegradable polyurethane is a synthetic biocompatible polymer material, is widely used in various biomedical fields because of its simple preparation method, high efficiency and superior biomedical properties, including drug slow control system and tissue engineering scaffold. Degrading polyesters or polycarbonate are endowed with degradability, but the degradation rate of these polyurethanes is slow and often fails to meet the requirements for the transfer of antitumor drugs. In order to improve the degradation rate of traditional polyurethane, researchers have some environmental responsive chemical bonds (such as pH sensitive ketal bond, acetal bond, hydrazone bond and reduction sensitive two sulfur). Bonds, etc., are introduced into the main chain of polyurethane. As a kind of acid sensitive chemical bond, the original ester bond has been widely used in anti-tumor nano drug carriers. However, the preparation of pH sensitive polyurethane and its application in the field of anti-tumor nanoscale drug carriers have not yet been reported. The two amino monomers and two kinds of polyhexyl glycol (Fw=530 and 2000) active esters with different molecular weights were polycondensation under mild conditions to obtain 2 pH sensitive poly (POEU1 and POEU2), and nanoparticles (NP1 and NP2) with the rate of inhomogeneous degradation were prepared by the monolayer emulsion of water oil (O/W). The structure and molecular weight of poly (tryptophone) were detected by gel permeation chromatography (GPC). The results showed that the structure of POEU1 and POEU2 was correct. The number of molecular weights (Mn) were 2.2 x 104 and 4.09104. transmission electron microscopy (TEM) and dynamic light diffractometer (DLS). The results showed that the particle size distribution of two nanoparticles was around 200 nm and all were spherical. The results show that the two nanoparticles are very stable under the physiological conditions (pH 7.4), while under the weak acid condition (pH 5), the degradation rate and the particle size decrease continuously. In addition, the degradation rate of the two nanoparticles (NP1 and NP2) can be controlled by the hydrophobicity of the polymer under the condition of pH 5. After 192 h, the weak hydrophobic POEU1 is prepared. The nanoparticles (NP1) were completely degraded, and the nanoparticles (NP2), prepared by the hydrophobic POEU2, only degraded the nanoparticles (NP1 and NP2) prepared by POEU1 and POEU2 (NP1 and NP2), which could efficiently encapsulate the antitumor drug adriamycin (DOX). Two kinds of drug loaded nanoparticles were obtained, named as NP1/DOX and NP2/DOX. drug release experimental nodes, respectively. The results showed that NP1/DOX and NP2/DOX significantly accelerated the drug release behavior, and the release rate could be regulated by the hydrophobicity of the polymer. In pH 5, the release rate of NP2/DOX prepared by the hydrophobic POEU2 was slower than the NP1/DOX. which was prepared by the relatively weak hydrophobic POEU1 through the stimulated confocal scanning microscope (CLSM) and the flow cells. The uptake of NP1/DOX and NP2/DOX by human neuroblastoma cells (SH-SY5Y) and mouse derived hepatoma cells (H22) was detected and quantified. The results showed that NP1/DOX and NP2/DOX could be effectively absorbed and released by SH-SY5Y and H22 cells and released adriamycin into the nucleus. The MTT method was used to detect NP1 and NP2 and drug carrier particles by MTT method. The survival rates of NP1/DOX and NP2/DOX with SH-SY5Y and H22 cells were co cultured for 24 h. The results showed that the survival rate of the cells was more than 90% for the blank nanoparticles, even at the maximum concentration of 1mg/mL, indicating that the blank nanoparticles NP1 and NP2 were basically non-toxic. However, NP1/DOX and NP2/ DOX could effectively kill cancer cell SH-SY5Y and cells. There is a concentration dependent killing effect, that is, the survival rate of SH-SY5Y and H22 cells decreases with the increase of the concentration of drug particles. The 3 dimensional multicellular ball (3D SH-SY5Y MCTS) formed by the human neuroblastoma cells is used as an in vitro tumor model to simulate the infiltration and growth inhibition of NP1/DOX and NP2/DOX to the tumor. The results show that with NP1, with NP1 The co culture time of /DOX and NP2/DOX and SH-SY5Y MCTS was prolonged, compared with the naked drug (DOX), NP1/DOX and NP2/DOX showed better permeability and higher growth inhibition to SH-SY5Y MCTS. The mouse model of liver cancer, established by mouse hepatoma cell H22, was used to detect two kinds of drug loaded nanoparticles NP1/DOX and NP2/DOX in the organs and swelling in the body. The distribution, side effects, and antitumor effects of the tumor tissue. The drug distribution in vivo shows that adriamycin, which is loaded in the nanoparticles, has longer blood circulation time, more effective tumor enrichment and lower cardiac toxicity compared with the naked drug (DOX). In vivo antitumor results show that within 7 days of the mice treated, the physiological saline The mice tumor in the group and the blank nanoparticles group (NP1 and NP2) increased continuously, and the tumor volume increased by 8-9 times in 7 days. However, the tumor in the naked drug (DOX) group and the two carrier group (NP1/DOX and NP2/DOX) were obviously suppressed, and the inhibition rate of NP1/DOX and NP2/DOX (72.68% and 70.37%) was higher than that of the naked drug (62.61%) group (62.61%). Finally, the tissue was used in the tissue. The slicing technique further assessed the side effects and antitumor effects of NP1/DOX and NP2/DOX. The results showed that the number of tumor tissue cells in the naked drug (DOX) group and the two drug loaded particles (NP1/DOX and NP2/DOX) mice decreased significantly and had varying degrees of necrosis compared to the saline group, and this phenomenon was given in NP1/DOX and NP2/DOX. In addition, the mice in the DOX group had serious heart necrosis. However, the hearts of the NP1/DOX and NP2/DOX mice were basically normal and no other organ toxicity. In conclusion, the good biocompatible pH sensitive poly (tryptophone) has a good application prospect in the field of antitumor drug delivery.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5
【相似文献】
相关期刊论文 前2条
1 陶扬洋;巩凯;王睿;唐汝培;;新型聚原酸酯共聚物的合成及性能研究[J];高分子通报;2013年06期
2 ;[J];;年期
相关会议论文 前4条
1 成静;杜福胜;冀然;李子臣;;一种合成含原酸酯侧基酸敏感高分子材料的药物载体新方法[A];2011年全国高分子学术论文报告会论文摘要集[C];2011年
2 宋成程;杜福胜;李子臣;;氧化反应促进含原酸酯酸敏感聚合物的降解[A];2013年全国高分子学术论文报告会论文摘要集——主题F：功能高分子[C];2013年
3 黄潇楠;杜福胜;李子臣;;含原酸酯侧基的酸/温度双敏感性(甲基)丙烯酰胺聚合物[A];中国化学会第26届学术年会功能高分子科学前沿分会场论文集[C];2008年
4 乔增莹;黄潇楠;杜福胜;李子臣;;用于药物载体的温度/酸双响应的聚合物胶束及囊泡[A];2011年全国高分子学术论文报告会论文摘要集[C];2011年
相关博士学位论文 前1条
1 闫国卿;pH超敏感聚原酸酯药物载体的设计、制备及抗肿瘤活性研究[D];安徽大学;2017年
相关硕士学位论文 前2条
1 陶扬洋;新型聚原酸酯的设计、合成及性能研究[D];江南大学;2013年
2 李相鹏;利用葡萄糖原酸酯区域选择性保护羟基的研究以及神经节苷脂GM3三糖的高效合成研究[D];中国海洋大学;2010年
，本文编号：2151726
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2151726.html
