多靶点脑靶向仿生纳米递药系统的构建及抗胶质瘤作用与机制研究
发布时间：2018-08-04 20:15
【摘要】：脑胶质瘤是中枢神经系统最常见的颅脑内肿瘤,发生于神经外胚层,世界卫生组织数据显示,脑胶质瘤是35岁以下中青年第2号肿瘤杀手,每年全球约有18~60万中青年被恶性脑胶质瘤夺去生命。胶质瘤体呈强侵袭性生长特点,外科手术不易分开肿瘤组织和正常组织,导致手术治疗效果不佳。术后的放化疗策略对于脑胶质瘤的治疗具有非常重要的意义。但是由于血脑屏障(blood brain barrier,BBB)的生理屏障作用,使得化疗药物难以透过BBB,进而到达肿瘤组织起到杀伤肿瘤细胞的作用。经特异性配体修饰的靶向纳米递药系统可降低化疗药物的副作用,增加药物在病变部位的蓄积,因此在肿瘤治疗领域具有重要意义。目前大多数纳米载体均为人工合成,作为外源性物质具有免疫原性。仿生型纳米递药系统是一种天然微粒系统,可以模拟体内物质或其他病原体,具有较低的免疫原性和良好的靶向性。仿生纳米递药系统可以分为三大类,分别是基于细胞、内源性蛋白和病原体的递药系统。以脂蛋白为基础构建的仿生型纳米给药系统是其中重要的一类,具有良好的生物相容性,同时其结构中的靶向配体,可与受体结合,具有天然的靶向能力,从而可获得良好的靶向作用。胶质瘤干细胞(glioma stem cells,GSCs)是脑胶质瘤中具有自我更新和多向分化潜能的细胞,该细胞与肿瘤的侵袭性生长、复发、耐药性有关。GSCs的存在使胶质瘤对传统的放疗和化疗产生耐受,并且保持着自我更新与增殖,诱导胶质瘤的复发。因此针对GSCs进行肿瘤的靶向治疗具有重要意义。盐霉素(salinomycin,SAL)被报道可以选择性杀伤多种肿瘤干细胞,同时有研究表明盐霉素对肿瘤细胞也有很好的杀伤作用。本课题构建一种靶向性的仿生纳米载体包载盐霉素,生物相容性良好,通过脑靶向功能肽Angiopep-2和LDL共同介导透过BBB进入脑部,靶向于胶质瘤表面的LDL和LRP-1受体,实现双极靶向递药,杀伤胶质瘤细胞和胶质瘤干细胞,同时抑制胶质瘤干细胞来源的血管内皮细胞,阻断肿瘤新生组织的血液供应,实现多靶点抗胶质瘤作用。课题第一部分,采用两种胶质瘤细胞系SHG-44和U87细胞,采用无血清悬浮培养基从胶质瘤细胞中分离培养出悬浮的细胞球,称为肿瘤微球体(tumorsphere,TS),并通过免疫荧光法验证两种肿瘤微球体均表达胶质瘤干细胞标记物CD133和巢蛋白(Nestin);流式细胞法检测CD133阳性率,SHG-44 TS中阳性细胞百分比41.48%,u87ts中阳性细胞百分比17.3%,和文献报道胶质瘤干细胞生长特性和特异性标记物的表达情况一致,表明无血清培养基方法得到的肿瘤微球体即为胶质瘤干细胞。而后分别考察sal对两种胶质瘤细胞系和gscs的毒性作用,结果表明sal对胶质瘤细胞和gscs均具有杀伤作用,但shg-44ts和u87ts对sal更为敏感。课题第二部分,从人血液中提取分离得到低密度脂蛋白(lowdensitylipoprotein,ldl),通过傅里叶红外光谱对其进行鉴定,bca法测得ldl的浓度为1.67mg/ml。将angiopep-2肽修饰于ldl上得到ldl-angiopep-2(ldl-ang),介导其跨越bbb,通过核心重组法制备载sal的ldl纳米粒,即ldl-sal-ang。对处方进行优化,包封率为35.63±2.65%,载药量为5.83±0.7%,粒径和电位分别为22.95±1.58nm和-5.98±0.20mv。课题第三部分,考察载盐霉素低密度脂蛋白纳米粒体外抗肿瘤细胞和肿瘤干细胞活性,激光共聚焦显微镜观察胶质瘤细胞和gscs对载荧光探针dii纳米粒(ldl-sal-dii-ang和ldl-sal-dii)的摄取情况。胶质瘤细胞和gscs对ldl-sal-dii-ang的摄取强度均高于ldl-sal-dii,并存在受体的竞争性抑制,证明ldl的核心包载sal和表面angiopep-2的连接不影响纳米粒与ldlr的结合。细胞毒性试验证明ldl和ldl-ang对细胞毒性较低。考察ldl-sal-ang和ldl-sal对胶质瘤细胞和gscs的毒性和诱导细胞凋亡情况。两种纳米粒对胶质瘤干细胞的毒性均强于sal,其中ldl-sal-ang具有最强细胞毒性作用。在shg-44细胞、u87细胞、shg-44ts和u87ts中,ldl-sal-ang的ic50值分别为0.17μm,3.66μm,0.16μm和0.04μm,同时诱导胶质瘤细胞及胶质瘤干细胞凋亡能力最强。考察纳米粒对胶质瘤细胞及gscs微球体形成的影响试验中,ldl-sal-ang组形成的微球数量最少,且球体疏松,说明纳米粒在较低药物浓度下就可抑制胶质瘤干细胞的自我更新。培养胶质瘤干细胞来源的血管内皮细胞,免疫荧光法进行鉴定,观察纳米粒对细胞小管形成能力和迁移能力的影响,结果表明ldl-sal-ang对细胞的小管形成和迁移抑制作用最为显著,说明ldl纳米粒通过作用于胶质瘤干细胞对其新生血管生成产生抑制作用。课题第四部分,考察了载盐霉素低密度脂蛋白纳米粒的脑转运特性。考察人脑微血管内皮细胞(humanbrainmicrovesselendothelialcell,hbmec)对ldl-dii-ang和ldl-dii的摄取情况,hbmec对纳米粒的摄取强度随时间延长而增强。采用transwell小室共培养模型,考察纳米粒在hbmec单层细胞的渗透情况,模拟其透bbb作用,结果表明ldl-dii-ang渗透率优于ldl-dii,达到80%。同时考察了纳米粒被transwell小室下室胶质瘤细胞及gscs所摄取情况,结果表明ldl纳米粒经Angiopep-2修饰后,增强跨BBB能力,提高细胞摄取,增强了抗肿瘤活性。制备载荧光探针DiR的纳米粒(LDL-DiR-Ang和LDL-Di R)。在裸鼠脑尾状核注射胶质瘤细胞悬液,制备裸鼠荷原位脑胶质瘤模型。动物活体成像观察载DiR纳米粒在小鼠体内的分布。与LDL-DiR相比,LDL-Di R-Ang在脑部聚集更多,荧光更强,具有良好的脑靶向性。同时荷原位瘤裸鼠尾静脉注射LDL-DiI-Ang和LDL-DiI,取荷瘤脑组织冰冻切片,激光共聚焦显微镜下观察,结果与活体成像一致。取ICR小鼠,空白LDL和LDL-Ang连续给药七天,取各组织进行CD68免疫组化染色,验证纳米粒无明显组织毒性。课题第五部分,进行载盐霉素低密度脂蛋白纳米粒的体内药代动力学和组织分布研究。将ICR小鼠分为三组,SAL、LDL-SAL和LDL-SAL-Ang组,观察不同组的SAL血药浓度随时间变化的情况和组织分布的情况。结果表明LDL-SAL和LDL-SAL-Ang均具有一定缓释作用,半衰期显著延长,药时曲线下面积显著增加。LDL-SAL-Ang在脑组织中的蓄积高于其他两组,脑组织对其摄取也显著高于其他两组,证明LDL-SAL-Ang具有较强的脑靶向能力。课题第六部分,考察载盐霉素低密度脂蛋白纳米粒的体内抗肿瘤活性。首先制备裸鼠荷原位脑胶质瘤模型,将纳米粒与一线抗胶质瘤药物替莫唑胺(TMZ)联合给药,给药组分为8组:对照组、SAL组、TMZ组、LDL-SAL组、LDL-SAL-Ang组、SAL+TMZ组、LDL-SAL+TMZ组和LDL-SAL-Ang+TMZ组。给药结束后脑组织切片行免疫组化染色,观察肿瘤部位胶质瘤细胞凋亡情况和血管新生情况,观察每组剩余小鼠生存状态和体重变化情况。结果表明LDL-SAL-Ang可促进原位脑胶质瘤中胶质瘤细胞凋亡并抑制血管新生,改善小鼠的生存状态,延长生存时间。给药组小鼠生存时间和状态均有延长和改善,其中LDL-SAL-Ang+TMZ组小鼠中位生存期长达47天,相对于对照组延长67.8%,体重下降最为缓慢,同时肿瘤细胞凋亡显著,新生血管数量最少,在给药组中显示最优的治疗效果,表明LDL-SAL-Ang具有体内抗肿瘤活性,与TMZ联用效果更佳,可有效治疗脑胶质瘤。以上研究结果表明,本课题构建的脑靶向给药系统LDL-SAL-Ang在体内外具有较强的抗胶质瘤干细胞作用,同时具有双极脑靶向特性,透过BBB后靶向于肿瘤组织,实现多靶点抗脑胶质瘤作用,为脑胶质瘤的化疗提供新思路和实验基础。
[Abstract]:Glioma is the most common Craniocerebral Tumor in the central nervous system and occurs in the neuroectoderm. The WHO data show that glioma is the No. second tumor killer of young and middle-aged people under 35 years of age. Every year, about 18~60 million of young people worldwide are killed by malignant glioma. The glioma is characterized by strong invasive growth and surgery is not easy. Apart from tumor tissue and normal tissue, the effect of surgical treatment is not good. The postoperative radiotherapy and chemotherapy strategy is of great significance for the treatment of glioma. But because of the physiological barrier of blood brain barrier (BBB), it makes the chemotherapeutic drugs difficult to penetrate into the tumor tissue to kill the tumor cells by BBB. The targeted nano drug delivery system modified by specific ligands can reduce the side effects of chemotherapeutic drugs and increase the accumulation of drugs in the lesion site. Therefore, it is of great significance in the field of tumor treatment. Most of the nanoscale carriers are synthesized by artificial synthesis and have immunogenicity as a exogenous substance. The bionic nano delivery system is a one. The natural particle system, which can simulate substance or other pathogens in the body, has a lower immunogenicity and good targeting. The bionic nano drug delivery system can be divided into three major categories, which are based on cell, endogenous protein and pathogen delivery system. The biomimetic nano drug delivery system based on lipoprotein is important. A class of glioma stem cells (GSCs) is a cell with self renewal and multidirectional differentiation potential in glioma, and the cell and tumor invasiveness. Growth, recurrence, and drug resistance related to the presence of.GSCs make glioma tolerant to traditional radiotherapy and chemotherapy, and maintain self renewal and proliferation and induce recurrence of glioma. Therefore, targeted therapy for GSCs is of great significance. Salinomycin (SAL) has been reported to be able to selectively kill a variety of tumor stem cells. At the same time, some studies have shown that salt mycin also has a good killing effect on tumor cells. This topic constructs a targeted biomimetic nanoscale carrier containing salt mycin, which has good biocompatibility, through the brain targeting functional peptide Angiopep-2 and LDL into the brain through BBB, targeting the LDL and LRP-1 receptors on the surface of the gelatin tumor to achieve bipolar targeting. Drug delivery, killing glioma cells and glioma stem cells, simultaneously inhibiting the vascular endothelial cells derived from glioma stem cells, blocking the blood supply of neoplastic tissue, and achieving the anti glioma effect of multiple targets. In the first part, two glioma cell lines, SHG-44 and U87 cells, were used in the serum-free suspension culture medium from glioma cells. The cell spheres were isolated and cultured in the medium, called tumorsphere (TS). By immunofluorescence, two kinds of tumor microspheres were demonstrated to express the markers CD133 and nestin (Nestin) of glioma stem cells. The positive rate of CD133 was detected by flow cytometry, the percentage of positive cells in SHG-44 TS and the percentage of positive cells in u87ts were 17.3%. The growth characteristics of glioma stem cells and the expression of specific markers were consistent with the literature. It showed that the tumor microspheres obtained by the serum-free medium were glioma stem cells. Then the toxic effects of SAL on two glioma cell lines and GSCs were examined respectively. The results showed that Sal had a killing effect on glioma cells and GSCs. But shg-44ts and u87ts are more sensitive to sal. In the second part, we extract and separate low density lipoprotein (lowdensitylipoprotein, LDL) from human blood and identify them by Fourier transform infrared spectroscopy. The concentration of LDL is 1.67mg/ml. by BCA method, and angiopep-2 peptide is trimed on LDL to get ldl-angiopep-2 (ldl-ang), which is mediated by 1.67mg/ml.. The preparation of SAL loaded LDL nanoparticles, namely ldl-sal-ang., was optimized by the core recombination method, the encapsulation efficiency was 35.63 + 2.65%, the drug loading was 5.83 + 0.7%, the particle size and potential were 22.95 + 1.58nm and -5.98 + 0.20mv., respectively, and the activity of anti tumor cells and tumor stem cells in vitro of low density lipoprotein nanoparticles was investigated. The uptake of DiI nanoparticles (ldl-sal-dii-ang and ldl-sal-dii) by glioma cells and GSCs was observed by laser confocal microscopy. The uptake of ldl-sal-dii-ang in glioma cells and GSCs was higher than that of ldl-sal-dii, and there was a competitive inhibition of the receptor, which proved that the connection between Sal and surface angiopep-2 of LDL was not connected to LDL. The combination of nanoparticles and LDLR. Cytotoxicity tests show that LDL and ldl-ang have low cytotoxicity. The toxicity of ldl-sal-ang and ldl-sal to glioma cells and GSCs and the induction of cell apoptosis. The toxicity of two nanoparticles to glioma stem cells is stronger than that of Sal, and ldl-sal-ang has the strongest cytotoxic effect. In the cells, U87 cells, shg-44ts and u87ts, the IC50 values of ldl-sal-ang were 0.17 mu m, 3.66 m, 0.16 mu m and 0.04 micron respectively. At the same time, the apoptosis ability of glioma cells and glioma stem cells was the strongest. It is indicated that nanoparticles can inhibit the self renewal of glioma stem cells at low drug concentration. The culture of vascular endothelial cells derived from glioma stem cells, identification of immunofluorescence, and the effect of nanoparticles on the formation and migration of cell tubules are observed. The results show that ldl-sal-ang has an inhibitory effect on the formation and migration of tubules. In the fourth part, the brain transport properties of low density lipoprotein nanoparticles (humanbrainmicrovesselendothelialcell, HBMEC) of human brain microvascular endothelial cells (humanbrainmicrovesselendothelialcell, HBMEC) were investigated in the fourth part of the study. The uptake of HBMEC nanoparticles increased with time. The Transwell cell co culture model was used to investigate the permeability of the nanoparticles in HBMEC monolayer cells, and the BBB effect was simulated. The results showed that the ldl-dii-ang permeability was better than that of ldl-dii, and the nanoparticle was detected by 80%. in the Transwell small chamber glioma at the same time. The results of cell and GSCs uptake showed that the LDL nanoparticles were modified by Angiopep-2 to enhance the ability to cross the BBB, improve the cell uptake and enhance the anti-tumor activity. The nanoparticles (LDL-DiR-Ang and LDL-Di R) loaded with the fluorescent probe DiR (LDL-DiR-Ang and LDL-Di R) were prepared in the nude mouse caudate nucleus by injection of glioma cell suspension to prepare the nude mouse in situ glioma model. The distribution of DiR nanoparticles in mice was observed by imaging. Compared with LDL-DiR, LDL-Di R-Ang had more aggregation in the brain, stronger fluorescence and better brain targeting. At the same time, LDL-DiI-Ang and LDL-DiI were injected into the tail vein of the nude mice. The frozen section of the tumor bearing brain tissue was taken under the laser confocal microscope, and the results were in accordance with the living body imaging. ICR mice, blank LDL and LDL-Ang were continuously given for seven days, and each tissue was stained by CD68 immunohistochemical staining to verify that the nanoparticles had no obvious histotoxicity. The fifth part of the subject was to study the pharmacokinetics and tissue distribution of the low density lipoprotein nanoparticles in the body, and the ICR mice were divided into three groups, SAL, LDL-SAL and LDL-SAL-Ang. The blood concentration of SAL in different groups was changed with time and tissue distribution. The results showed that both LDL-SAL and LDL-SAL-Ang had a sustained release effect, the half-life was prolonged significantly, the area under the curve of the drug time increased significantly and the accumulation of.LDL-SAL-Ang in the brain tissue was higher than that of the other two groups, and the uptake of the brain tissue was significantly higher than that of the other two groups. LDL-SAL-Ang has strong brain targeting ability. In the sixth part, the antitumor activity of low density lipoprotein nanoparticles was investigated in the body. First, the nude mice bearing in situ glioma model was prepared, and the nanoparticles were combined with the first line antiglioma drug temozolomide (TMZ), and the drug group was divided into 8 groups: the control group, the SAL group, the TMZ group, and the LDL-SA. Group L, group LDL-SAL-Ang, group SAL+TMZ, group LDL-SAL+TMZ, and LDL-SAL-Ang+TMZ group. After the end of the administration, the brain tissue sections were immunohistochemical staining, the apoptosis and angiogenesis of glioma cells at the tumor site were observed, and the survival and weight changes of the remaining mice in each group were observed. The results showed that LDL-SAL-Ang could promote the in situ glioma in situ. The apoptosis of glioma cells and inhibition of angiogenesis, improve the survival state of mice and prolong the survival time. The survival time and state of the mice were prolonged and improved. The median survival period of the LDL-SAL-Ang+TMZ group was 47 days longer than that of the control group, which was 67.8% and the decrease was the slowness, and the apoptosis of the tumor cells was significant. The number of blood vessels is the least, which shows the best therapeutic effect in the drug delivery group. It shows that LDL-SAL-Ang has antitumor activity in the body and better combined with TMZ, which can effectively treat glioma. The above results show that the brain targeting drug delivery system, which is constructed by the project, has a strong anti glioma stem cell effect in vivo and in vivo and in vitro, and has a good effect on LDL-SAL-Ang. It has the characteristics of bipolar brain targeting, which targets the tumor tissue through BBB, and achieves multiple targets against glioma. It provides a new idea and experimental basis for the chemotherapy of brain glioma.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943
本文编号：2165015
[Abstract]:Glioma is the most common Craniocerebral Tumor in the central nervous system and occurs in the neuroectoderm. The WHO data show that glioma is the No. second tumor killer of young and middle-aged people under 35 years of age. Every year, about 18~60 million of young people worldwide are killed by malignant glioma. The glioma is characterized by strong invasive growth and surgery is not easy. Apart from tumor tissue and normal tissue, the effect of surgical treatment is not good. The postoperative radiotherapy and chemotherapy strategy is of great significance for the treatment of glioma. But because of the physiological barrier of blood brain barrier (BBB), it makes the chemotherapeutic drugs difficult to penetrate into the tumor tissue to kill the tumor cells by BBB. The targeted nano drug delivery system modified by specific ligands can reduce the side effects of chemotherapeutic drugs and increase the accumulation of drugs in the lesion site. Therefore, it is of great significance in the field of tumor treatment. Most of the nanoscale carriers are synthesized by artificial synthesis and have immunogenicity as a exogenous substance. The bionic nano delivery system is a one. The natural particle system, which can simulate substance or other pathogens in the body, has a lower immunogenicity and good targeting. The bionic nano drug delivery system can be divided into three major categories, which are based on cell, endogenous protein and pathogen delivery system. The biomimetic nano drug delivery system based on lipoprotein is important. A class of glioma stem cells (GSCs) is a cell with self renewal and multidirectional differentiation potential in glioma, and the cell and tumor invasiveness. Growth, recurrence, and drug resistance related to the presence of.GSCs make glioma tolerant to traditional radiotherapy and chemotherapy, and maintain self renewal and proliferation and induce recurrence of glioma. Therefore, targeted therapy for GSCs is of great significance. Salinomycin (SAL) has been reported to be able to selectively kill a variety of tumor stem cells. At the same time, some studies have shown that salt mycin also has a good killing effect on tumor cells. This topic constructs a targeted biomimetic nanoscale carrier containing salt mycin, which has good biocompatibility, through the brain targeting functional peptide Angiopep-2 and LDL into the brain through BBB, targeting the LDL and LRP-1 receptors on the surface of the gelatin tumor to achieve bipolar targeting. Drug delivery, killing glioma cells and glioma stem cells, simultaneously inhibiting the vascular endothelial cells derived from glioma stem cells, blocking the blood supply of neoplastic tissue, and achieving the anti glioma effect of multiple targets. In the first part, two glioma cell lines, SHG-44 and U87 cells, were used in the serum-free suspension culture medium from glioma cells. The cell spheres were isolated and cultured in the medium, called tumorsphere (TS). By immunofluorescence, two kinds of tumor microspheres were demonstrated to express the markers CD133 and nestin (Nestin) of glioma stem cells. The positive rate of CD133 was detected by flow cytometry, the percentage of positive cells in SHG-44 TS and the percentage of positive cells in u87ts were 17.3%. The growth characteristics of glioma stem cells and the expression of specific markers were consistent with the literature. It showed that the tumor microspheres obtained by the serum-free medium were glioma stem cells. Then the toxic effects of SAL on two glioma cell lines and GSCs were examined respectively. The results showed that Sal had a killing effect on glioma cells and GSCs. But shg-44ts and u87ts are more sensitive to sal. In the second part, we extract and separate low density lipoprotein (lowdensitylipoprotein, LDL) from human blood and identify them by Fourier transform infrared spectroscopy. The concentration of LDL is 1.67mg/ml. by BCA method, and angiopep-2 peptide is trimed on LDL to get ldl-angiopep-2 (ldl-ang), which is mediated by 1.67mg/ml.. The preparation of SAL loaded LDL nanoparticles, namely ldl-sal-ang., was optimized by the core recombination method, the encapsulation efficiency was 35.63 + 2.65%, the drug loading was 5.83 + 0.7%, the particle size and potential were 22.95 + 1.58nm and -5.98 + 0.20mv., respectively, and the activity of anti tumor cells and tumor stem cells in vitro of low density lipoprotein nanoparticles was investigated. The uptake of DiI nanoparticles (ldl-sal-dii-ang and ldl-sal-dii) by glioma cells and GSCs was observed by laser confocal microscopy. The uptake of ldl-sal-dii-ang in glioma cells and GSCs was higher than that of ldl-sal-dii, and there was a competitive inhibition of the receptor, which proved that the connection between Sal and surface angiopep-2 of LDL was not connected to LDL. The combination of nanoparticles and LDLR. Cytotoxicity tests show that LDL and ldl-ang have low cytotoxicity. The toxicity of ldl-sal-ang and ldl-sal to glioma cells and GSCs and the induction of cell apoptosis. The toxicity of two nanoparticles to glioma stem cells is stronger than that of Sal, and ldl-sal-ang has the strongest cytotoxic effect. In the cells, U87 cells, shg-44ts and u87ts, the IC50 values of ldl-sal-ang were 0.17 mu m, 3.66 m, 0.16 mu m and 0.04 micron respectively. At the same time, the apoptosis ability of glioma cells and glioma stem cells was the strongest. It is indicated that nanoparticles can inhibit the self renewal of glioma stem cells at low drug concentration. The culture of vascular endothelial cells derived from glioma stem cells, identification of immunofluorescence, and the effect of nanoparticles on the formation and migration of cell tubules are observed. The results show that ldl-sal-ang has an inhibitory effect on the formation and migration of tubules. In the fourth part, the brain transport properties of low density lipoprotein nanoparticles (humanbrainmicrovesselendothelialcell, HBMEC) of human brain microvascular endothelial cells (humanbrainmicrovesselendothelialcell, HBMEC) were investigated in the fourth part of the study. The uptake of HBMEC nanoparticles increased with time. The Transwell cell co culture model was used to investigate the permeability of the nanoparticles in HBMEC monolayer cells, and the BBB effect was simulated. The results showed that the ldl-dii-ang permeability was better than that of ldl-dii, and the nanoparticle was detected by 80%. in the Transwell small chamber glioma at the same time. The results of cell and GSCs uptake showed that the LDL nanoparticles were modified by Angiopep-2 to enhance the ability to cross the BBB, improve the cell uptake and enhance the anti-tumor activity. The nanoparticles (LDL-DiR-Ang and LDL-Di R) loaded with the fluorescent probe DiR (LDL-DiR-Ang and LDL-Di R) were prepared in the nude mouse caudate nucleus by injection of glioma cell suspension to prepare the nude mouse in situ glioma model. The distribution of DiR nanoparticles in mice was observed by imaging. Compared with LDL-DiR, LDL-Di R-Ang had more aggregation in the brain, stronger fluorescence and better brain targeting. At the same time, LDL-DiI-Ang and LDL-DiI were injected into the tail vein of the nude mice. The frozen section of the tumor bearing brain tissue was taken under the laser confocal microscope, and the results were in accordance with the living body imaging. ICR mice, blank LDL and LDL-Ang were continuously given for seven days, and each tissue was stained by CD68 immunohistochemical staining to verify that the nanoparticles had no obvious histotoxicity. The fifth part of the subject was to study the pharmacokinetics and tissue distribution of the low density lipoprotein nanoparticles in the body, and the ICR mice were divided into three groups, SAL, LDL-SAL and LDL-SAL-Ang. The blood concentration of SAL in different groups was changed with time and tissue distribution. The results showed that both LDL-SAL and LDL-SAL-Ang had a sustained release effect, the half-life was prolonged significantly, the area under the curve of the drug time increased significantly and the accumulation of.LDL-SAL-Ang in the brain tissue was higher than that of the other two groups, and the uptake of the brain tissue was significantly higher than that of the other two groups. LDL-SAL-Ang has strong brain targeting ability. In the sixth part, the antitumor activity of low density lipoprotein nanoparticles was investigated in the body. First, the nude mice bearing in situ glioma model was prepared, and the nanoparticles were combined with the first line antiglioma drug temozolomide (TMZ), and the drug group was divided into 8 groups: the control group, the SAL group, the TMZ group, and the LDL-SA. Group L, group LDL-SAL-Ang, group SAL+TMZ, group LDL-SAL+TMZ, and LDL-SAL-Ang+TMZ group. After the end of the administration, the brain tissue sections were immunohistochemical staining, the apoptosis and angiogenesis of glioma cells at the tumor site were observed, and the survival and weight changes of the remaining mice in each group were observed. The results showed that LDL-SAL-Ang could promote the in situ glioma in situ. The apoptosis of glioma cells and inhibition of angiogenesis, improve the survival state of mice and prolong the survival time. The survival time and state of the mice were prolonged and improved. The median survival period of the LDL-SAL-Ang+TMZ group was 47 days longer than that of the control group, which was 67.8% and the decrease was the slowness, and the apoptosis of the tumor cells was significant. The number of blood vessels is the least, which shows the best therapeutic effect in the drug delivery group. It shows that LDL-SAL-Ang has antitumor activity in the body and better combined with TMZ, which can effectively treat glioma. The above results show that the brain targeting drug delivery system, which is constructed by the project, has a strong anti glioma stem cell effect in vivo and in vivo and in vitro, and has a good effect on LDL-SAL-Ang. It has the characteristics of bipolar brain targeting, which targets the tumor tissue through BBB, and achieves multiple targets against glioma. It provides a new idea and experimental basis for the chemotherapy of brain glioma.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943
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