pH响应性磁靶向阿霉素复合纳米载体的构建与应用研究
发布时间：2019-05-22 19:46
【摘要】：恶性肿瘤的发病率逐年上升,是严重威胁人类健康的重大疾病。化疗是目前临床最常用的肿瘤治疗手段之一。但由于一般化疗药物对肿瘤细胞和组织不具有选择性,导致广泛的毒副作用,严重影响了患者的生活质量并限制了化疗药物的临床应用。因此,设计新型靶向肿瘤的药物载体系统,提高化疗药物体内对肿瘤部位的靶向性,在治疗过程中使化疗药物能够在肿瘤部位靶向富集,有效提高肿瘤组织的药物浓度,减小全身性的毒副作用,成为当前肿瘤给药系统研究的热点。本研究中我们拟采用效果明确的磁靶向技术为靶向策略,根据肿瘤部位pH低于正常组织的生理特点,设计合成能够通过静电作用有效结合化疗药物阿霉素(DOX)药物分子且具有pH敏感释放特性的高分子聚合物与磁性纳米粒构建形成阿霉素的肿瘤靶向性控释复合纳米给药体系。以期在外磁场作用下,该化疗药物载体在体内能够定向富集于肿瘤病灶部位,在肿瘤组织较低pH的生理条件下有效释放负载的化疗药物,增加药物在肿瘤部位的浓度,降低化疗药物的全身毒性,从而达到更好的治疗效果,提高患者的顺应性。在第一部分纳米药物载体的构建、合成和表征中,我们首先合成了pH响应性的聚合物载药材料聚乙二醇-b-聚天冬氨酸(PEG-b-Pasp),反应首先是由L-天冬氨酸-β-苄酯与无水四氢呋喃,在60℃和三光气的环境中反应制备得到天冬氨酸苄酯环内酸酐,再与氨基聚乙二醇单甲醚发生开环聚合反应得到聚乙二醇-b-聚天冬氨酸苄酯(PEG-b-PBLA),最后通过在OH-条件下水解去除PEG-b-PBLA的苄醇,经过核磁共振和红外吸收光谱表征表明成功合成了PEG-b-PAsp。然后利用水热法合成了粒径水合粒径约140nm的四氧化三铁(Fe_3O_4)磁性纳米粒,为提高Fe_3O_4磁性纳米粒在空气中的稳定性并降低其相互聚集,我们通过St?ber方法和溶胶凝胶过程在Fe_3O_4磁性纳米粒表面包覆上一层厚度约10nm二氧化硅(SiO_2)层,最后通过酰胺化反应将合成的PEG-b-PAsp与表面氨基化的SiO_2层化学交联,再加入盐酸阿霉素溶液搅拌过夜进行载药,制备出Fe_3O_4@SiO_2@PEG-b-PAsp@DOX纳米复合载体。经对该纳米复合载体的粒径、Zeta电位、载药量等的表征,结果表明制备得到的纳米复合载体为球状,粒径较均一,约(197.7±1.5)nm,Zeta电位约为(-35.9±0.6)mv,DOX的载药量约20.36%。在pH分别为5.5和7.4的缓冲溶液中的药物释放实验表明该纳米复合载药体系具有阿霉素pH敏感性释放的特点。第二部分我们考察了载药纳米粒在体外磁场作用下的细胞靶向性,并考察了该纳米复合载体对小鼠肺癌细胞LLC杀伤作用和载药材料的初步安全性。荧光显微镜和生物透射电镜观察表明,载体材料能够以内吞的方式被肿瘤细胞有效摄取并聚集于肿瘤细胞中。在同一细胞培养体系中加入5μg·m L~(-1)的Fe_3O_4@SiO_2@PEG-b-PAsp@DOX载药体系共培养2h后,利用流式细胞仪借助DOX的红色荧光对载药体系的体外靶向性进行了定量检测,在其施加外界磁场一侧的细胞中荧光强度(Xmean=98.65)显著强于未施加外界磁场一侧的细胞的荧光强度(Xmean=81.81),两者之间存在显著性差异(p0.05),表明该载药体系在外界磁场下有较显著的靶向性,而且在进入肿瘤细胞后DOX能够有效被释放。在该载药纳米复合载体杀伤肿瘤细胞作用研究中发现,载药复合纳米粒可以有效的杀伤LLC细胞;通过CCK-8法测定结果计算出24h时游离DOX和载药的纳米复合载体的IC50值分别为0.46μg·m L~(-1)和0.89μg·m L~(-1),在48h时两者的IC50分别为0.32μg·m L~(-1)和0.46μg·m L~(-1),可知随着时间的延长载药纳米复合载体杀伤肿瘤细胞能力逐渐接近游离DOX,这可能纳米载药体系中DOX的缓释有关。我们还利用CCK-8法和流式细胞仪初步考察了空白载体的生物安全性,在空白载体浓度高达100μg·m L~(-1)时,细胞与空白材料共培养48h后,三种不同的细胞(LLC细胞珠、人的肺癌细胞A549细胞株和成纤维细胞L929细胞株)存活率均在80%以上,而较低浓度的空白载体具有较好的生物安全性,几乎不存在细胞毒性。流式检测结果也说明,空白载体几乎不会诱导肿瘤细胞的凋亡,而载药后的复合纳米材料可以有效诱导肿瘤细胞凋亡的发生,凋亡的比例随载药浓度的增加而增加,其作用与游离DOX对肿瘤细胞的作用一致。第三部分我们利用所建立的LLC细胞荷瘤小鼠模型研究了纳米复合载药体系在体内的靶向性和抗肿瘤效果。通过在C57小鼠的右后腿外侧皮下注射LLC细胞悬液建立荷瘤小鼠模型。在考察纳米复合载药体系的体内靶向性实验中,荷瘤小鼠随机分为施加和未施加外界磁场组,尾静脉注射4mg·kg~(-1)的纳米复合载药体系溶液后,施加磁场组在肿瘤部位固定磁铁4h,然后分别将各组肿瘤部位置于3.0 T磁共振成像仪中进行T2加权成像检测,结果表明与对照相比施加磁场组肿瘤部位的铁顺磁性物质富集明显大于未施加磁场组,二者差别有统计学意义(p=0.0040.01),表明该纳米复合载药体系在小鼠体内具有良好的磁靶向富集特性。在纳米复合载药体系体内抗肿瘤效果研究中,我们将荷瘤小鼠分为生理盐水组(S)、游离DOX组(FD)、未施加磁场组(P)、施加磁场组(MP)四组,通过记录各组小鼠的生存率、肿瘤体积、体重的变化和肺转移结节数考察纳米复合载药体系的抗肿瘤效果。在每组5只的荷瘤小鼠存活实验中,第36天各组的生存率分别为40%(S)、20%(FD)、80%(P)和100%(MP);肿瘤增长曲线表明生理盐水组和未施加磁场组小鼠肿瘤生长稍快;而在小鼠体重生长曲线中可以明显看到只有游离DOX组小鼠的平均体重随着治疗的进行呈下降趋势,结合小鼠存活实验和肿瘤增长实验的结果,说明在该浓度下游离DOX直接给药可能产生较大的系统毒性。通过对模型小鼠解剖的观察,发现纳米复合载药体系可抑制模型小鼠肿瘤的肺部转移,其机制有待于进一步研究。本研究成功构建的pH响应性的磁靶向复合纳米载药体系能够有效的负载DOX,并表现出显著的药物pH响应释放和磁靶向性,在体内外均显示出良好的抗肿瘤作用,有望在外磁场作用下靶向的到达肿瘤病灶部位,实现在肿瘤部位可控释药,减小抗肿瘤药物化疗副作用,为新型靶向抗肿瘤药物传递系统的研究提供了有益的探索。
[Abstract]:The incidence of malignant tumor is increasing year by year, which is a serious disease which is a serious threat to human health. Chemotherapy is one of the most common methods for the treatment of cancer. But because the general chemotherapy drugs do not have the selectivity to the tumor cells and the tissues, the invention leads to a wide toxic and side effect, and the quality of the life of the patients is seriously affected and the clinical application of the chemotherapy medicine is limited. therefore, the novel drug carrier system for targeting the tumor is designed, the targeting property of the tumor part in the chemotherapy medicine body is improved, the chemotherapy medicine can be targeted and enriched at the tumor part in the treatment course, the drug concentration of the tumor tissue is effectively improved, the systemic toxic and side effect is reduced, And is a hot spot for the research of the current tumor drug delivery system. In the study, we intend to use the magnetic targeting technology with definite effect as the targeting strategy, and according to the physiological characteristics of the tumor site, the pH is lower than that of the normal tissue, In that design and synthesis, the adriamycin (DOX) drug molecule and the high-molecular polymer with the pH-sensitive release characteristic and the magnetic nano-particle can be effectively combined with the chemotherapeutic drug adriamycin (DOX) drug molecule to form the adriamycin-based tumor-targeted controlled-release composite nano-drug delivery system. so as to effectively release the loaded chemotherapy drug under the physiological condition of lower pH of the tumor tissue, increase the concentration of the drug at the tumor site and reduce the systemic toxicity of the chemotherapeutic drug, So as to achieve better treatment effect and improve the compliance of the patient. In the construction, synthesis and characterization of the first part of the nano-drug carrier, we first synthesized the pH-responsive polymer drug-carrying material, polyethylene glycol-b-polyaspartic acid (PEG-b-Pasp), and the reaction is first carried out by the L-aspartic acid-1-phthalate and the anhydrous four-hydrogen peroxide, reacting in an environment of 60 DEG C and triphosgene to obtain an aspartic ester ring-b-polyaspartic acid ester (PEG-b-PBLA) in an open-loop polymerization reaction with the amino-polyethylene glycol monomethyl ether, and finally, performing hydrolysis to remove the polyoxyalkylene of the PEG-b-PBLA under the OH-condition, The results showed that PEG-b-PASP was successfully synthesized by nuclear magnetic resonance and infrared absorption spectrum. then the ferroferric oxide (Fe _ 3O _ 4) magnetic nano-particles with a particle size of about 140 nm are synthesized by a hydrothermal method, so that the stability of the Fe _ 3O _ 4 magnetic nano-particles in the air is improved, in that sol-gel process, a layer of silicon dioxide (SiO _ 2) with a thickness of about 10 nm is coat on the surface of the Fe _ 3O _ 4 magnetic nano-particle by a St-ber method and a sol-gel process, The Fe_3O_4@SiO_2 @ PEG-b-PAsp@DOX nano composite carrier is prepared. The results of the characterization of the particle size, Zeta potential and drug-carrying amount of the nano-composite carrier show that the prepared nano-composite carrier is spherical, the particle size is uniform, about (197.7-1.5) nm, the Zeta potential is about (-35.9-0.6) mv, and the loading amount of DOX is about 20.36%. The drug release experiment in the buffer solution with pH of 5.5 and 7.4 shows that the nano-composite drug-carrying system has the characteristics of the adriamycin pH-sensitive release. In the second part, we investigated the cell targeting of the drug-loaded nanoparticles under the action of the in vitro magnetic field, and examined the anti-killing effect of the nano-composite carrier on the mouse lung cancer cell LLC and the initial safety of the drug-loaded material. The observation of the fluorescence microscope and the transmission electron microscope shows that the carrier material can be taken up by the tumor cells and accumulated in the tumor cells in a manner that the carrier material can be swallowed. In the same cell culture system,5. mu. g. m L-(-1) of the Fe_3O_4@SiO_2 @ PEG-b-PAsp@DOX drug-carrying system was co-cultured for 2 h, and the in vitro targeting of the drug-carrying system was quantitatively detected by flow cytometry using the red fluorescence of DOX. The fluorescence intensity (Xmean = 98.65) in the cells on the side of which the external magnetic field was applied was significantly stronger than that of the cells without the external magnetic field (Xmean = 81.81), and there was a significant difference between the two (p0.05), indicating that the drug-carrying system had significant targeting at the external magnetic field. And DOX can be effectively released after entering the tumor cells. in that study of the effect of the drug-loaded nano-composite carrier on the tumor cell, the drug-loaded composite nanoparticle can effectively kill the LLC cell, and the IC50 value of the free DOX and the drug-loaded nano composite carrier at 24 h is 0.46. mu. g 路 m L-(-1) and 0.89. m The IC50 of the two was 0.32 & mu; g 路 m-L ~ (-1) and 0.46. m u.g 路 m-L ~ (-1) respectively at 48 h, and it was found that the ability of the drug-loaded nano-composite carrier to kill the tumor cells gradually approaches the free DOX over time, which may be related to the slow release of DOX in the nano-drug-loaded system. We also used CCK-8 method and flow cytometer to study the biological safety of blank carrier, and when the concentration of blank carrier was as high as 100 & mu; g 路 m L ~ (-1), the cells were co-cultured with blank material for 48 h, and three different cells (LLC cell beads, The survival rate of the human lung cancer cell A549 cell line and the fibroblast cell line L929 cell line is more than 80%, and the lower concentration blank carrier has better biological safety, and has little cytotoxicity. The results of the flow test also show that the blank carrier can hardly induce the apoptosis of the tumor cells, and the compound nano-materials after the loading can effectively induce the apoptosis of the tumor cells, and the proportion of the apoptosis is increased with the increase of the concentration of the drug carrier, and the effect is consistent with the effect of the free DOX on the tumor cells. In the third part, we study the targeting and anti-tumor effect of the nano-composite drug-loaded system in vivo using the established model of LLC cell-bearing mice. The tumor-bearing mouse model was established by subcutaneous injection of the LLC cell suspension on the right hind leg of the C57 mouse. in the in vivo targeting experiment of the nano composite drug-carrying system, the tumor-bearing mice were randomly divided into the nano-composite drug-carrying system solution applied and not applied to the external magnetic field group and the tail vein injection of 4 mg/ kg to (-1), and the magnetic field group is applied to the tumor part to fix the magnet 4h, Then, the tumor sites of each group were placed in a 3.0T magnetic resonance imager for T2-weighted imaging, and the results showed that the concentration of the iron paramagnetic substance in the tumor part of the magnetic field group was significantly higher than that of the non-applied magnetic field group compared with the control group (p = 0.0040.01). It is shown that the nano-composite drug-loaded system has good magnetic targeting and enrichment characteristics in mice. in that study of the anti-tumor effect of the nano composite drug-loaded system, the tumor-bearing mice are divided into a normal saline group (S), a free DOX group (FD), an unapplied magnetic field group (P), a magnetic field group (MP) group, The anti-tumor effect of the nano-composite drug-loaded system was investigated by the change of body weight and the number of lung metastases. The survival rate of tumor-bearing mice in each group was 40% (S),20% (FD),80% (P) and 100% (MP), respectively. In the mouse body weight growth curve, the average body weight of only free DOX group mice was seen to decrease with the treatment, and combined with the results of the mouse survival experiment and the tumor growth experiment, it was shown that the free DOX direct administration at this concentration could result in a greater systemic toxicity. Through the observation of the model mouse, it is found that the nano-composite drug-loading system can inhibit the lung metastasis of the model mouse tumor, and its mechanism is to be further studied. The successfully constructed pH-responsive magnetic-targeting composite nano-drug-loading system is capable of effectively loading DOX, and shows remarkable drug pH response release and magnetic targeting, and has good anti-tumor effect in vivo. Can be targeted at the site of the tumor focus under the action of the external magnetic field, realize the controlled release medicine at the tumor site, reduce the side effect of the anti-tumor drug chemotherapy, and provide a beneficial exploration for the research of a novel targeting anti-tumor drug delivery system.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943
本文编号：2483203
[Abstract]:The incidence of malignant tumor is increasing year by year, which is a serious disease which is a serious threat to human health. Chemotherapy is one of the most common methods for the treatment of cancer. But because the general chemotherapy drugs do not have the selectivity to the tumor cells and the tissues, the invention leads to a wide toxic and side effect, and the quality of the life of the patients is seriously affected and the clinical application of the chemotherapy medicine is limited. therefore, the novel drug carrier system for targeting the tumor is designed, the targeting property of the tumor part in the chemotherapy medicine body is improved, the chemotherapy medicine can be targeted and enriched at the tumor part in the treatment course, the drug concentration of the tumor tissue is effectively improved, the systemic toxic and side effect is reduced, And is a hot spot for the research of the current tumor drug delivery system. In the study, we intend to use the magnetic targeting technology with definite effect as the targeting strategy, and according to the physiological characteristics of the tumor site, the pH is lower than that of the normal tissue, In that design and synthesis, the adriamycin (DOX) drug molecule and the high-molecular polymer with the pH-sensitive release characteristic and the magnetic nano-particle can be effectively combined with the chemotherapeutic drug adriamycin (DOX) drug molecule to form the adriamycin-based tumor-targeted controlled-release composite nano-drug delivery system. so as to effectively release the loaded chemotherapy drug under the physiological condition of lower pH of the tumor tissue, increase the concentration of the drug at the tumor site and reduce the systemic toxicity of the chemotherapeutic drug, So as to achieve better treatment effect and improve the compliance of the patient. In the construction, synthesis and characterization of the first part of the nano-drug carrier, we first synthesized the pH-responsive polymer drug-carrying material, polyethylene glycol-b-polyaspartic acid (PEG-b-Pasp), and the reaction is first carried out by the L-aspartic acid-1-phthalate and the anhydrous four-hydrogen peroxide, reacting in an environment of 60 DEG C and triphosgene to obtain an aspartic ester ring-b-polyaspartic acid ester (PEG-b-PBLA) in an open-loop polymerization reaction with the amino-polyethylene glycol monomethyl ether, and finally, performing hydrolysis to remove the polyoxyalkylene of the PEG-b-PBLA under the OH-condition, The results showed that PEG-b-PASP was successfully synthesized by nuclear magnetic resonance and infrared absorption spectrum. then the ferroferric oxide (Fe _ 3O _ 4) magnetic nano-particles with a particle size of about 140 nm are synthesized by a hydrothermal method, so that the stability of the Fe _ 3O _ 4 magnetic nano-particles in the air is improved, in that sol-gel process, a layer of silicon dioxide (SiO _ 2) with a thickness of about 10 nm is coat on the surface of the Fe _ 3O _ 4 magnetic nano-particle by a St-ber method and a sol-gel process, The Fe_3O_4@SiO_2 @ PEG-b-PAsp@DOX nano composite carrier is prepared. The results of the characterization of the particle size, Zeta potential and drug-carrying amount of the nano-composite carrier show that the prepared nano-composite carrier is spherical, the particle size is uniform, about (197.7-1.5) nm, the Zeta potential is about (-35.9-0.6) mv, and the loading amount of DOX is about 20.36%. The drug release experiment in the buffer solution with pH of 5.5 and 7.4 shows that the nano-composite drug-carrying system has the characteristics of the adriamycin pH-sensitive release. In the second part, we investigated the cell targeting of the drug-loaded nanoparticles under the action of the in vitro magnetic field, and examined the anti-killing effect of the nano-composite carrier on the mouse lung cancer cell LLC and the initial safety of the drug-loaded material. The observation of the fluorescence microscope and the transmission electron microscope shows that the carrier material can be taken up by the tumor cells and accumulated in the tumor cells in a manner that the carrier material can be swallowed. In the same cell culture system,5. mu. g. m L-(-1) of the Fe_3O_4@SiO_2 @ PEG-b-PAsp@DOX drug-carrying system was co-cultured for 2 h, and the in vitro targeting of the drug-carrying system was quantitatively detected by flow cytometry using the red fluorescence of DOX. The fluorescence intensity (Xmean = 98.65) in the cells on the side of which the external magnetic field was applied was significantly stronger than that of the cells without the external magnetic field (Xmean = 81.81), and there was a significant difference between the two (p0.05), indicating that the drug-carrying system had significant targeting at the external magnetic field. And DOX can be effectively released after entering the tumor cells. in that study of the effect of the drug-loaded nano-composite carrier on the tumor cell, the drug-loaded composite nanoparticle can effectively kill the LLC cell, and the IC50 value of the free DOX and the drug-loaded nano composite carrier at 24 h is 0.46. mu. g 路 m L-(-1) and 0.89. m The IC50 of the two was 0.32 & mu; g 路 m-L ~ (-1) and 0.46. m u.g 路 m-L ~ (-1) respectively at 48 h, and it was found that the ability of the drug-loaded nano-composite carrier to kill the tumor cells gradually approaches the free DOX over time, which may be related to the slow release of DOX in the nano-drug-loaded system. We also used CCK-8 method and flow cytometer to study the biological safety of blank carrier, and when the concentration of blank carrier was as high as 100 & mu; g 路 m L ~ (-1), the cells were co-cultured with blank material for 48 h, and three different cells (LLC cell beads, The survival rate of the human lung cancer cell A549 cell line and the fibroblast cell line L929 cell line is more than 80%, and the lower concentration blank carrier has better biological safety, and has little cytotoxicity. The results of the flow test also show that the blank carrier can hardly induce the apoptosis of the tumor cells, and the compound nano-materials after the loading can effectively induce the apoptosis of the tumor cells, and the proportion of the apoptosis is increased with the increase of the concentration of the drug carrier, and the effect is consistent with the effect of the free DOX on the tumor cells. In the third part, we study the targeting and anti-tumor effect of the nano-composite drug-loaded system in vivo using the established model of LLC cell-bearing mice. The tumor-bearing mouse model was established by subcutaneous injection of the LLC cell suspension on the right hind leg of the C57 mouse. in the in vivo targeting experiment of the nano composite drug-carrying system, the tumor-bearing mice were randomly divided into the nano-composite drug-carrying system solution applied and not applied to the external magnetic field group and the tail vein injection of 4 mg/ kg to (-1), and the magnetic field group is applied to the tumor part to fix the magnet 4h, Then, the tumor sites of each group were placed in a 3.0T magnetic resonance imager for T2-weighted imaging, and the results showed that the concentration of the iron paramagnetic substance in the tumor part of the magnetic field group was significantly higher than that of the non-applied magnetic field group compared with the control group (p = 0.0040.01). It is shown that the nano-composite drug-loaded system has good magnetic targeting and enrichment characteristics in mice. in that study of the anti-tumor effect of the nano composite drug-loaded system, the tumor-bearing mice are divided into a normal saline group (S), a free DOX group (FD), an unapplied magnetic field group (P), a magnetic field group (MP) group, The anti-tumor effect of the nano-composite drug-loaded system was investigated by the change of body weight and the number of lung metastases. The survival rate of tumor-bearing mice in each group was 40% (S),20% (FD),80% (P) and 100% (MP), respectively. In the mouse body weight growth curve, the average body weight of only free DOX group mice was seen to decrease with the treatment, and combined with the results of the mouse survival experiment and the tumor growth experiment, it was shown that the free DOX direct administration at this concentration could result in a greater systemic toxicity. Through the observation of the model mouse, it is found that the nano-composite drug-loading system can inhibit the lung metastasis of the model mouse tumor, and its mechanism is to be further studied. The successfully constructed pH-responsive magnetic-targeting composite nano-drug-loading system is capable of effectively loading DOX, and shows remarkable drug pH response release and magnetic targeting, and has good anti-tumor effect in vivo. Can be targeted at the site of the tumor focus under the action of the external magnetic field, realize the controlled release medicine at the tumor site, reduce the side effect of the anti-tumor drug chemotherapy, and provide a beneficial exploration for the research of a novel targeting anti-tumor drug delivery system.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943
【参考文献】
相关期刊论文 前3条
1 Ahmet Bilici;;Treatment options in patients with metastatic gastric cancer:Current status and future perspectives[J];World Journal of Gastroenterology;2014年14期
2 朱杰;廖蕾;朱丽娜;孔继烈;刘宝红;;叶酸功能化介孔碳纳米球负载阿霉素的细胞靶向传递及可控释放[J];化学学报;2013年01期
3 刘再毅;王瑛;王广谊;李晓红;李晏;粱长虹;;超顺磁性氧化铁标记脂肪干细胞移植入大鼠心脏后的活体磁共振示踪成像[J];中国医学科学院学报;2009年02期
，本文编号：2483203
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2483203.html
最近更新
教材专著
