多肽介导的脑胶质瘤靶向递药策略研究

发布时间：2018-06-21 23:50

本文选题：脑胶质瘤 + 化疗　；参考：《复旦大学》2014年硕士论文

【摘要】：脑胶质瘤是中枢神经系统最为常见的肿瘤,占脑部肿瘤发病数40%左右。脑胶质瘤多生长在脑部重要部分,比如中央沟区、丘脑及脑干等区域,因此于术难度大,而目.脑胶质瘤侵润性高,瘤体细胞生长速度快,通过手术无法将肿瘤组织完全清除。化疗是目前治疗神经上皮肿瘤和改善预后的重要手段之一。然而化疗药物对脑胶质瘤患者的体内疗效却不甚理想。其主要原因可以归结为以下几个方面：(1)化疗药物无选择性,组织分布广泛,对肿瘤组织无靶向作用,毒副作用大；(2)由胶质瘤新生血管内皮细胞和胶质瘤细胞所组成的血瘤屏障(blood-brain tumor barrier, BBTB)的存在极大的限制了化疗药物向胶质瘤部位的的递送；(3)脑胶质瘤属于实体肿瘤,化疗药物对脑胶质瘤的穿透性差；(4)细胞外基质(ECM)是脑胶质瘤的重要组成部分,占脑胶质瘤微环境的80%以上,细胞外基质在胶质瘤体的形态维持、胶质瘤细胞的分化和增殖起到重要作用,但目前针对脑胶质瘤细胞外基质的治疗手段却鲜见报道。因此,构建高效主动靶向、增加肿瘤穿透和以细胞外基质为靶点的递药策略有望提高脑胶质瘤的化疗效果,具有较高研究价值和临床意义。针对脑胶质瘤的生理和病理学特征,本课题采用不同的靶向策略以期增加化疗药物对脑胶质瘤的治疗效果。本文第一部分设计了CGKRK多肽修饰的纳米递药系统,其能同时靶向胶质瘤新生血管内皮细胞和胶质瘤细胞,增加化疗药物在脑胶质瘤部位的蓄积；第二部分构建了肿瘤渗透促进多肽tLyp-1修饰的纳米递药系统,在靶向BBTB的同时,增加了化疗药物在血管以及肿瘤实体的穿透能力；第三部分设计了内化型多肽F3修饰的纳米递药系统联合肿瘤渗透多肽tLyp-1共给药策略,进一步提高化疗药物在肿瘤部位的蓄积以及穿透；第四部分以肿瘤细胞外基质和肿瘤新生血管为靶点,构建了CGKRK和ATWLPPR双重修饰的双级靶向系统,使其能同时靶向肿瘤微环境和新生血管,提高治疗效果。本文第一部分设计了CGKRK多肽修饰的双重靶向递药系统。CGKRK多肽的受体是硫酸乙酰肝素,该受体高表达于胶质瘤新生血管内皮细胞和胶质瘤细胞表面,而在正常组织细胞中不表达,具有很强的特异性。因此以CGKRK多肽修饰的纳米递药系统可以双重靶向于肿瘤新生血管和胶质瘤细胞,增加化疗药物在肿瘤部位的蓄积。这部分研究中首先通过开环聚合法合成纳米材料聚乙二醇聚己内酯(MPEG-PCL)和马来酰亚胺聚乙二醇聚己内酯(Maleimide-PEG-PCL)并采用乳化溶媒蒸发法制备包载PTX的纳米粒(NP-PTX),利用马来酰亚胺基和巯基的特异性反应构建CGKRK修饰的载紫杉醇纳米粒(CGKRK-NP-PTX)。制得的CGKRK-NP-PTX大小均匀,形态圆整,平均粒径为117 nm, Zeta电位为-15.7 mV。细胞摄取结果显示,与普通纳米粒相比,CGKRK修饰的纳米粒显著增加了HUVEC细胞和U87MG细胞的摄取,且在这两种细胞上的摄取为浓度、时间和温度依赖。摄取抑制实验显示,CGKRK-NP在HUVEC细胞上的内吞需要能量,通过细胞膜穴样凹陷和脂筏介导的两种内吞途径入胞,并且内吞过程有微管蛋白的参与；在U87MG细胞上的内吞需要能量,通过细胞膜穴样凹陷和脂筏介导的两种内吞途径入胞,并且内吞过程有高尔基体的参与。U87MG肿瘤球渗透实验和生长抑制实验表明CGKRK修饰的纳米递药系统在增加肿瘤球穿透能力的同时显著抑制了肿瘤球的生长。体外诱导凋亡实验和毒性实验显示,经过CGKRK多肽修饰后,载PTX纳米粒的体外诱导HUVEC细胞和U87MG细胞凋亡的能力以及毒性都有显著性增高。小动物活体成像和脑组织的冰冻切片结果显不,CGKRK肽修饰的纳米粒在肿瘤部位的荧光强度明显高于未修饰的纳米粒,表明CGKRK肽的修饰显著提高了纳米粒透过肿瘤血管的能力,增加了肿瘤部位的蓄积。荷皮下U87MG的裸鼠肿瘤抑制实验显示,在给药后,经过CGKRK多肽修饰的载PTX纳米粒的抗实体瘤生长能力最强。本文第二部分构建了肿瘤渗透促进多肽tLyp-1修饰的纳米递药系统,tLyp-1多肽的受体是肿瘤新生血管和肿瘤细胞上均高表达的神经纤毛蛋白Neuropilin-1(NRP-1)蛋白,NRP-1蛋白是血管上的转膜蛋白,能介导肿瘤血管内外的物质运输。本部分将tLyp-1多肽修饰在纳米粒表面,以期靶向肿瘤新生血管和肿瘤细胞的同时,增加纳米粒从血管中的渗透以及肿瘤穿透。用乳化溶媒蒸发法制备包载PTX的PEG-PLA纳米粒(NP-PTX),并通过马来酰亚胺基团和巯基的特异性反应将tLyp-1多肽共价结合到纳米粒表面。制备的tLyp-1-NP-PTX粒径在111nm左右,Zeta电位-24.3 mV. HUVEC细胞和C6细胞定性定量结果显示, HUVEC细胞和C6细胞对tLyp-1-NP的摄取呈现时间、浓度和温度依赖性,且显著高于未修饰NP组。摄取抑制实验结果表明tLyp-1-NP的内吞需要能量,在HUVEC上的摄取主要通过陷穴小泡和脂筏介导的两种内吞途径入胞,并且内吞过程有高尔基体和酸性溶酶体参与,在C6细胞上的摄取主要通过脂筏介导的,并且由高尔基体参与。通过C6细胞毒和细胞凋亡等实验表明tLyp-1肽修饰的纳米粒显著增大了PTX对胶质瘤细胞的抑制作用。体外C6细胞三维肿瘤球模型评价结果表明tLyp-1肽的修饰显著增强了纳米粒对实体肿瘤的渗透能力和抑制能力。通过荷C6原位瘤小鼠的体内实验评价tLyp-1修饰的纳米递药系统的胶质瘤体内靶向性。小动物活体成像和脑组织的冰冻切片结果显示,tLyp-1肽的修饰显著提高了纳米粒穿过肿瘤血管的能力和在胶质瘤部位的蓄积。原位胶质瘤裸鼠的生存时间考察结果显示,tLyp-1-NP-PTX组的平均生存时间为37天,显著高于Saline组(18天) (p0.001,), Taxol组(23天)(p0.001), NP-PTX组(28天)(p0.01),表明载PTX纳米粒经tLyp-1肽修饰后具有更强的抗脑胶质瘤作用。本文第三部分以内化型多肽F3修饰纳米递药系统联合肿瘤渗透促进多肽tLyp-1构建共给药纳米递释系统。F3肽的受体是肿瘤新生血管和肿瘤细胞上高表达的核仁素,且核仁素不在正常细胞表面,随着肿瘤细胞的恶化程度增加而表达量增加,是肿瘤靶向给药的理想靶点。tLyp-1是一种具有碳端规则((R/K)XX(R/K))的多肽,可以特异性靶向NRP-1受体,激活一种特殊的血管运输系统,同时增加组织渗透。本部分研究将F3肽修饰的PEG-PLA纳米粒联合tLyp-1肽共同注射构建共给药系统,以克服BTB的阻碍作用,进一步增加纳米粒透过胶质瘤血管的能力和在肿瘤部位的蓄积。制得F3-NP粒径在125 nm左右,zeta电位为-13.3 mV,并通过透射电镜和表面元素分析对其表征。以C6细胞为模型,细胞摄取实验结果表明与未修饰的NP相比,F3-NP可以通过筏介导内吞以及能量依赖的直接转运途径极大地提高细胞摄取量,并且是高尔基体参与的过程。体外C6细胞三维肿瘤球模型评价结果表明F3肽的修饰显著增强了纳米粒对实体肿瘤的渗透能力。小动物活体成像和脑组织的冰冻切片结果显示,F3肽的修饰以及tLyp-1的共给药策略显著提高了纳米粒穿过肿瘤血管的能力和在胶质瘤部位的蓄积。原位胶质瘤裸鼠的生存时间考察结果显示,F3-NP-PTX+tLyp-1组的平均生存时间为42天,显著高于Saline组(19天)(p0.001),Taxol组(24天)(p 0.001), NP-PTX组(28天)(p0.001),F3-NP组(32天)(p0.05)和F3-NP-PTX+tLyp-1组(32天)(p0.01),表明载紫杉醇纳米粒经F3肽修饰和tLyp-1多肽共给药后具有更强的抗脑胶质瘤作用。本文的第四个部分选取细胞外基质和肿瘤新生血管为靶点,构建了CGKRK多肽和ATWLPPR共修饰的双级靶向系统.CGKRK多肽的受体是硫酸乙酰肝素,而ATWLPPR多肽的受体是NRP-1。硫酸乙酰肝素是细胞外基质的重要组成成分,与肿瘤的复发再生以及转移具有很大关系。采用半胱氨酸基团将ATWLPPR多肽和CGKRK多肽相连接,形成ATWLPPRCCGKRK (AC)多肽,通过马来酰亚胺基团的特异性反应制得AC-NP,粒径在123 nm左右,zeta电位为-11.4 mV,透射电镜显示形态圆整,大小均一。HUVEC细胞和U87MG细胞模型评价结果显示经过AC联合肽修饰的纳米粒摄取显著增加,载PTX对U87MG细胞的诱导凋亡能力和毒性显著提高。AC-NP在HUVEC上的摄取是能量依赖的,由陷穴小泡和脂筏共同介导的内吞,并有酸性溶酶体的参与；AC-NP在U87MG上的摄取是能量依赖的,由陷穴小泡和脂筏共同介导的,有微管蛋白、高尔基体和酸性溶酶体的参与。体外U87MG细胞三维肿瘤球模型评价结果表明AC肽的修饰显著增强了纳米粒对实体肿瘤的渗透能力和生长抑制能力。小动物活体成像和脑组织的冰冻切片结果显示,AC肽修饰的纳米粒在脑胶质瘤部位的荧光强度明显高于未修饰的纳米粒以及ATWLPPR多肽和CGKRK多肽单修饰的纳米粒,显著.提高了纳米粒透过肿瘤血管的能力,增加了胶质瘤部位的蓄积。同时体内抗脑胶质瘤实验显示AC-NP-PTX组的平均生存时间为51天,显著高于Saline组(22天)(p0.001),Taxol组(29天)(p0.001),NP-PTX组(31天)(p0.001),ATWLPPR-NP-PTX组(41.5天)(p0.01)和CGKRK-NP-PTX组(42天)(p0.01),表明ATWLPPR多肽和CGKRK多肽双重修饰显著性提高了荷U87MG原位瘤裸鼠的中位生存期。综上所述,本研究针对脑胶质瘤的生理和病理学特征,设计了具有胶质瘤新生血管和胶质瘤细胞双重靶向效果、提高肿瘤穿透效率和靶向细胞外基质的递药系统,为脑胶质瘤的靶向化疗提供了实验和理论依据,具有一定参考价值。
[Abstract]:Glioma is the most common tumor in the central nervous system, accounting for about 40% of the brain tumors. The brain glioma grows in important parts of the brain, such as the central sulcus, thalamus and brain stem, so it is difficult, but the brain glioma has high invasion, the growth rate of the tumor cells is fast, and the tumor can not be completely organized through surgery. Chemotherapy is one of the important means to treat the neuroepithelial tumor and improve the prognosis. However, the therapeutic effect of chemotherapeutic drugs on the patients with glioma is not ideal. The main reasons can be attributed to the following aspects: (1) there is no selective chemotherapy drug, extensive tissue distribution, no target effect on tumor tissue, and toxic and side effects. (2) the presence of blood-brain tumor barrier (BBTB), composed of glioma neovascular endothelial cells and glioma cells, greatly restricts the delivery of chemotherapeutic drugs to glioma sites; (3) glioma is a solid tumor, and the penetration of chemotherapeutic drugs to glioma is poor; (4) the extracellular matrix (ECM) is the brain. The important part of glioma accounts for more than 80% of the microenvironment of glioma. The extracellular matrix is maintained in the form of glioma, and the differentiation and proliferation of glioma cells play an important role. However, the treatment methods for the extracellular matrix of glioma are rarely reported. Therefore, the efficient and active target is constructed to increase the penetration of the tumor and to increase the penetration of the tumor. The delivery strategy of extracellular matrix as a target is expected to improve the therapeutic effect of brain glioma. It has high research value and clinical significance. In view of the physiological and pathological features of glioma, this subject uses different targeting strategies to increase the therapeutic effect of chemotherapeutic drugs on glioma. The first part of this paper is designed to modify the CGKRK polypeptide. The nano drug delivery system can target glioma neovascular endothelial cells and glioma cells at the same time, increase the accumulation of chemotherapeutic drugs in the brain glioma, and the second part constructs the nano delivery system of tumor infiltration promoting peptide tLyp-1 modification, and increases the penetration of chemotherapeutic drugs in blood vessels and tumor entities at the same time as target BBTB. The third part designed an internalized polypeptide F3 modified nano drug delivery system combined with tumor osmotic polypeptide tLyp-1 co delivery strategy to further improve the accumulation and penetration of chemotherapeutic drugs in the tumor site. The fourth part was targeted by the tumor extracellular matrix and tumor neovascularization, and the dual modification of CGKRK and ATWLPPR was constructed. The first part of this paper has designed the CGKRK peptide modified double targeting delivery system.CGKRK polypeptide, which is highly expressed in the neovascular endothelial cells of glioma and the surface of glioma cells, and in normal tissues. The CGKRK peptide modified nano drug delivery system can double target in neovascularization and glioma cells, and increase the accumulation of chemotherapeutic drugs at the tumor site. In this part, the nanomaterial polyethylene glycol polyhexyl (MPEG-PCL) and Malay were first synthesized by ring opening polymerization. The PTX nanoparticles (NP-PTX) were prepared by the emulsion solvent evaporation method, and the CGKRK modified paclitaxel nanoparticles (CGKRK-NP-PTX) were constructed by the specific reaction of maleimide and sulfhydryl groups. The CGKRK-NP-PTX size was uniform and the shape was round, and the average particle size was 117 nm, Zeta The uptake of -15.7 mV. cells showed that CGKRK modified nanoparticles significantly increased the uptake of HUVEC and U87MG cells compared with ordinary nanoparticles, and the uptake on these two cells was concentration, time and temperature dependence. The uptake inhibition experiment showed that the endocytosis of CGKRK-NP on HUVEC cells required energy through the cell membrane. The two endocytic pathways mediated by depression and lipid rafts enter the cell, and the endocytosis process is involved in microtubule protein; the endocytosis on U87MG cells requires energy, two endocytic pathways mediated by cell membrane cavern and lipid rafts, and the endocytosis process has a Golgi body involvement in the.U87MG tumor sphere penetration experiment and growth inhibition experiment. The CGKRK modified nano drug delivery system significantly inhibited the growth of tumor balls while increasing the penetration ability of tumor balls. In vitro induction of apoptosis and toxicity experiments showed that after the modification of CGKRK peptide, the ability to induce apoptosis of HUVEC cells and U87MG cells with PTX nanoparticles in vitro increased significantly. The results of imaging and brain tissue frozen section showed that the fluorescence intensity of CGKRK peptide modified nanoparticles at the tumor site was significantly higher than that of unmodified nanoparticles, indicating that the modification of CGKRK peptide significantly increased the capacity of nanoparticles through the tumor vessels and increased the accumulation of tumor sites. After that, the anti solid tumor growth ability of the loaded PTX nanoparticles modified by CGKRK polypeptide is the strongest. The second part of this paper has constructed the nano delivery system of tumor infiltration promoting peptide tLyp-1 modification. The receptor of tLyp-1 polypeptide is the high expression of the tumor cells and the high expression of the Neuropilin-1 (NRP-1) protein of the tumor cells, and the NRP-1 protein is a protein. The transmembrane protein on the vessel can mediate the transport of substances inside and outside the tumor. This part modifies the tLyp-1 peptide on the surface of the nanoparticles to target the neovascularization and tumor cells of the tumor, and increase the penetration of the nanoparticles from the blood vessels and the penetration of the tumor. The PTX PEG-PLA nanoparticles (NP-PTX) are prepared by the emulsification solvent evaporation method. The tLyp-1 polypeptide was covalently bound to the nanoparticles by the specific reaction of the maleimide group and the sulfhydryl group. The prepared tLyp-1-NP-PTX particle size was around 111nm, and the Zeta potential -24.3 mV. HUVEC cells and C6 cells qualitatively and quantitatively showed that HUVEC and C6 cells showed the time, concentration and temperature dependence of the concentration and temperature dependence. The uptake inhibition test results showed that the endocytosis of tLyp-1-NP needed energy, and the uptake on HUVEC was mainly through two endocytic pathways mediated by trapping vesicles and lipid rafts, and the endocytosis process was involved with Golgi and acid lysosomes, and the uptake of C6 cells was mainly mediated by lipid rafts, and by Golgi. The effects of tLyp-1 peptide modified nanoparticles on the inhibitory effect of PTX on glioma cells were significantly increased by C6 cytotoxicity and cell apoptosis. The evaluation of three-dimensional tumor ball model of C6 cells in vitro showed that the modification of tLyp-1 peptide significantly enhanced the osmotic ability and inhibition ability of nanoparticles to solid tumors. By loading C6 in situ tumor In vivo experiments in mice evaluate the targeting of glioma in the tLyp-1 modified nano delivery system. The results of small animal living imaging and brain tissue section show that the modification of tLyp-1 peptide significantly improves the capacity of nanoparticles through the tumor vessels and accumulation in the site of glioma. The results of the survival time of the nude mice of the primary glioma The average survival time of group tLyp-1-NP-PTX was 37 days, significantly higher than that in group Saline (18 days) (p0.001,), group Taxol (23 days) and NP-PTX group (28 days) (P0.01), indicating that the PTX nanoparticles were modified with the tLyp-1 peptide to be more resistant to glioma. The third part of this article was the combination of the internalized polypeptide F3 modified nano delivery system and the tumor infiltration. The receptor of peptide tLyp-1 for the construction of the.F3 peptide of the co delivery system is a highly expressed nucleolus on the neovascularization and tumor cells of the tumor, and the nucleolus is not on the surface of the normal cell. With the increase of the tumor cell deterioration, the expression of the nucleolus is increased, which is an ideal target for targeting the tumor,.TLyp-1 is a kind of carbon end rule (R/ K) XX (R/K) peptide, which can specifically target NRP-1 receptor, activates a special vascular transport system and increases tissue permeability. This part studies the co injection system of F3 peptide modified PEG-PLA nanoparticles combined with tLyp-1 peptide to overcome BTB's hindrance and further increase the capacity of nanoparticles through the glioma vessels. The grain size of F3-NP was about 125 nm, and the zeta potential was -13.3 mV and characterized by transmission electron microscopy and surface element analysis. C6 cells were used as models. The results of cell uptake experiments showed that F3-NP could be greatly improved through raft mediated endocytosis and energy dependent direct transport, compared with unmodified NP. Cell uptake was the process of Golgi involvement. The evaluation of C6 cells in vitro showed that the modification of F3 peptide significantly enhanced the osmotic ability of the nanoparticles to solid tumors. The results of small animal living imaging and brain tissue frozen section showed that the modification of F3 peptide and the strategy of CO delivery of tLyp-1 increased significantly. The survival time of the nude mice in situ showed that the average survival time of the F3-NP-PTX+tLyp-1 group was 42 days, significantly higher than that in the Saline group (p0.001), Taxol (24 days) (P 0.001), NP-PTX group (28 days) (p0.001), F3-NP group (32 days) (P0.05) and F3-NP-PTX+tLyp-1 (P0.05) and F3-NP-PTX+tLyp-1. Group (32 days) (P0.01) (P0.01) showed that the paclitaxel nanoparticles had a stronger anti glioma effect after the F3 peptide modification and tLyp-1 polypeptide co administration. The fourth parts of this paper selected the extracellular matrix and tumor neovascularization as the target. The receptor of the CGKRK polypeptide and ATWLPPR modified double target system.CGKRK polypeptide was the liver sulfate acetyl liver. The receptor of ATWLPPR polypeptide is an important component of NRP-1. acetyl heparan sulfate, an important component of the extracellular matrix, which is closely related to the recurrence and metastasis of the tumor. Using the cysteine group, the ATWLPPR polypeptide and the CGKRK polypeptide are connected to form a ATWLPPRCCGKRK (AC) polypeptide, and the specific reaction of the maleimide group is made to produce AC. -NP, the particle size is about 123 nm, the zeta potential is -11.4 mV, the transmission electron microscope shows the shape round, the size of the.HUVEC cell and the U87MG cell model evaluation results show that the uptake of nanoparticles modified by AC combined peptide is significantly increased, and the ability to induce apoptosis and toxicity of U87MG cells is significantly increased by PTX, and the uptake of.AC-NP on HUVEC is energy dependent. The uptake of acupoint vesicles and lipid rafts co mediated endocytosis and the involvement of acid lysosomes; the uptake of AC-NP on U87MG is energy dependent, with the involvement of trapping vesicles and lipid rafts, with the involvement of microtubulin, Golgi bodies and acid lysosomes. The evaluation of the three dimensional tumor ball model in vitro shows that the modification of the AC peptide in vitro is significant. The results showed that the fluorescence intensity of AC peptide modified nanoparticles in brain glioma was significantly higher than that of unmodified nanoparticles as well as ATWLPPR polypeptide and CGKRK polypeptides modified nanoparticles. In vivo anti glioma experiments showed that the average survival time of AC-NP-PTX group was 51 days, significantly higher than group Saline (p0.001), group Taxol (29 days), NP-PTX group (31 days) (p0.001), ATWLPPR-NP-PTX group (41.5 days) (P0.01) and CGKRK-NP-PTX group (42 days) (P0.01). It shows that the double modification of ATWLPPR polypeptide and CGKRK polypeptide improves the median survival of nude mice bearing U87MG in situ tumor. To sum up, this study designed the dual targeting effect of glioma neovascularization and glioma cells to improve the penetration efficiency of tumor and target extracellular matrix against the physiological and pathological characteristics of glioma. Drug delivery system provides experimental and theoretical basis for targeted chemotherapy of glioma and has a certain reference value.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R96

【共引文献】