【作者】 王曦；

【导师】 滕利荣；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2019， 博士

【摘要】 脑胶质瘤具有生长迅速,复发率高和侵袭性强等特性,是目前死亡率最高的几类肿瘤之一。化学药物治疗是最常用的脑胶质瘤治疗手段,但是由于血脑屏障（BBB）的存在,大多数化疗药物难以穿透BBB递送进入大脑,并且药物对于肿瘤细胞不具有选择性,限制了化疗药物对脑胶质瘤的疗效。药物递送系统是解决以上问题的策略之一,其中又以具有低免疫原性、低毒性、生物可降解、高生物相容性等优点的脂质体最为适宜,脂质体表面含有丰富的官能团,能够对其改性、修饰后使其达到更优的性质,如主动靶向肿瘤细胞以及通过部分之前不能通过的生物屏障等。八聚精氨酸（R8）是一种典型的细胞穿膜肽（Cell-penetrating peptide,CPPs）,具有较强的细胞膜穿透能力和较强的阳离子特性,能够与细胞表面的负电残基形成氢键,在细胞膜电势作用下穿过细胞膜。而且R8富含氨基,可以通过酰胺键与疏水基团（如脂肪酸、油酸等）连接进行修饰。转铁蛋白（Tf）是一种糖蛋白,Tf作为脑靶向配体与TfRs具有较强的亲和力,Tf修饰赋予脂质体脑肿瘤靶向能力,递送化疗药物进入脑内达到靶向治疗脑胶质瘤的目的。基于以上研究背景,本文以化疗药物阿霉素（DOX）为模型药物,构建CPPs和Tf共修饰DOX脂质体,赋予其穿过BBB、肿瘤细胞双主动靶向及高效穿膜功能,首先,利用脂质体表面的Tf介导脂质体主动靶向BBB,当脂质体与BBB足够接近的情况下在CPPs的作用下,脂质体高效穿过BBB,然后,在脑内Tf介导脂质体主动靶向脑胶质瘤细胞,同样,CPPs可以介导脂质体穿过肿瘤细胞膜,最终,将DOX送达肿瘤细胞质。本文具体围绕DOX分析方法学的建立、CPPs修饰脂质体的处方筛选及工艺优化、性质表征、CPPs和Tf共修饰脂质体的制备及抗脑胶质瘤活性研究等方面展开研究。具体研究结果如下:1.DOX分析方法学的建立:为了对DOX含量进行定量分析,本章建立了DOX体内外分析方法学,并对方法学的各项性质进行考察。首先,使用高效液相色谱（HPLC）对DOX分析方法的精密度、回收率、稳定性进行研究,建立体外分析方法学。然后,建立超高效液相色谱串联质谱法（UPLC/MS/MS）检测脑组织中DOX含量的体内分析方法学,并对方法的准确度、精密度、回收率、基质效应和稳定性进行评价。结果显示,HPLC方法可以在体外准确、精密分析脂质体中DOX的含量,方法学满足DOX体外定量分析要求;UPLC/MS/MS分析方法可以准确、精密定量分析脑组织中DOX的含量,分析方法可以排除脑组织中生物基质对DOX检测的干扰。UPLC/MS/MS分析方法学满足DOX体内定量分析的要求。2.OA-R8脂质体递送DOX系统的建立:本章对CPPs修饰脂质体的处方和工艺进行优化,以其得到理化性质最优的脂质体进行下一步修饰。使用乙醇注入法结合硫酸铵梯度法主动载药制备CPPs修饰的DOX脂质体（R8PLP）,以粒径、包封率等作为评价指标对R8PLP中阳离子磷脂/OA-R8/ePC/胆固醇/DSPE-PEG2000之间的比例、阳离子磷脂种类、硫酸铵浓度及脂相/硫酸铵溶液的体积比等因素进行优化。制备R8PLP的最佳条件为DOTAP作为阳离子磷脂,DOTAP/OA-R8/ePC/Chol/DSPE-PEG=20/25/18/32/3,脂相/水相=1/10,硫酸铵浓度为250μM,搅拌速度为300 rpm,注入速度为5.5 mL/min制备R8PLP;DOX主动载药条件:选用药脂比为1/10,在pH值为7.4、温度45℃的条件下载药时间30 min,完成R8PLP的载药。3.OA-R8脂质体递送DOX系统的评价:本章主要对前文制备的R8PLP的体内外性质进行表征。首先,制备了用于脑部递送DOX的R8PLP,然后,对R8PLP的理化性质进行表征,体外抗肿瘤活性、细胞摄取进行相应评价。制备的R8PLP呈球形,表面光滑,分散性好,粒径均一,粒径大约为100 nm;R8PLP的ζ电位为14.71 mV;R8PLP在37℃和4℃条件下,在10.0%血清、PBS和去离子水中,48 h内稳定性良好;冻干后的R8PLP在37℃和4℃条件下,在10.0%血清、PBS和去离子水中,48 h内稳定性良好。DOX、LP、PLP、R8LP、R8PL对U87和GL261细胞具有显著的抗肿瘤活性,并且抗肿瘤活性具有时间依赖性;给药24 h后,R8LP、R8PL活性显著高于DOX、LP、PLP,推测OA-R8修饰可以提高DOX对U87和GL261细胞的抗肿瘤活性。最后,对R8PLP的BBB通过能力进行评价:与PLP相比,R8PLP在脑组织内蓄积效果显著,R8PLP中DOX在脑内的药时曲线下面积（AUC0.5-12h）为PLP的2.4倍。然而,R8PLP的半衰期(t_1/2)与PLP相似（3.47 vs 3.94 h）,这些结果表明,OA-R8修饰的脂质体具有更好的BBB穿透性和脑组织蓄积能力。4.Tf和OA-R8共修饰脂质体递送DOX系统的建立及评价:本章在R8PLP的基础上,讨论了Tf含量对脂质体靶向性与抗肿瘤效果的影响,建立了Tf和OA-R8共修饰脂质体递送DOX系统（Tf-R8PLP）,并对其粒径、血清中的稳定性、包封率和药物体外释放性能进行评价。除此之外,分析了Tf-R8PLP的体外细胞摄取。建立了裸鼠胶质瘤模型,并评估了Tf-R8PLP的体内抗肿瘤功效。对Tf-R8PLP治疗后,药物在裸鼠体内的组织分布和裸鼠器官（包括脑、心、脏、肝、脾、肺和肾）的病理学进行评价。Tf-R8PLP的最佳制备条件为R8PLP/Tf-PEG-DSPE的摩尔比例为1/100;Tf-R8PLP的粒径为128.64 nm,ξ-电位为6.81 mV,Tf的浓度为16.15μg/mL;Tf-R8PLP具有球形结构,粒径均一,Tf-R8PLP在血清中的稳定性良好;Tf-R8PLP均呈现了控制释放行为;Tf-R8PLP具有最高的肿瘤生长抑制率,Tf-R8PLP的体内抗肿瘤活性显著高于R8PLP和DOX;Tf-R8PLP组的小鼠生存时间（25天）均比生理盐水组（20天）、DOX组（22天）和R8PLP组（24天）长,Tf-R8PLP对于脑胶质瘤的治疗具有显著的效果。综上所述,本文制备了具有BBB、肿瘤细胞双主动靶向及高效穿膜功能的DOX脂质体,该脂质体理化性质优异,在体内外均有较好的抗肿瘤效果。为治疗脑胶质的多功能脂质体的临床应用打下理论基础。更多还原

【Abstract】 Glioma is characterized by rapid growth,high recurrence rate and strong invasiveness.Chemotherapy drugs are widely used in the course control of glioma,but due to the presence of the blood-brain barrier（BBB）,most of the chemotherapy drugs are difficult to penetrate BBB for brain delivery,and drugs for cancer cells may not be selective,they have a certain toxicity to normal cells,these factors limit the effect of chemotherapy drugs on brain glioma.Liposomes have the advantages of no immunogenicity,low toxicity,biodegradability and high biocompatibility,etc.The surface of liposomes contains abundant functional groups,which can improve the efficacy of liposomes in the treatment of tumors after modfied.Cell penetrating peptides（CPPs）are kinds of short peptides could improve drugs or biological macromolecules to cross the blood-brain barrier such as the intestinal wall,retina,neurons and other biological barriers to deliver.Octapolyarginine（R8）is a classic CPPs.It is characterized by strong cell membrane penetration and cationic properties and it is capable of hydrogen bonding with negative cell surface residues to cross the cell membrane under the action of membrane potential.In addition,R8 is rich in amino groups,which can be modified by bonding with hydrophobic groups（such as fatty acids,oleic acids,etc.）through amide bonds.Studies have shown that transferrin receptor（TfRs）in cerebral microvascular endothelial cells and glioma cells overexpressing,transferrin（Tf）is a kind of glycoprotein,Tf as brain targeting ligands with TfRs with strong affinity,Tf modified liposome give brain tumor targeting ability,delivering chemotherapy drugs into the brain to achieve the purpose of targeted therapy of cerebral glioma.Based on the above research background,this paper mainly focused on the establishment of doxorubicin（DOX）analytical methodology,formulation optimization of CPPs modified liposomes,characterization of liposomes properties,preparation of CPPs and Tf co-modified liposomes,and research on anti-glioma activity.Specific research results are as follows:1.Establishment of doxorubicin assay methodology:（1）High performance liquid chromatography（HPLC）was used to study the precision,recovery and stability of DOX assay method,and to establish in vitro assay methodology.（2）An in vivo analysis method for determination of DOX content in brain tissues by UPLC/MS/MS was established and the accuracy,precision,recovery,matrix effect and stability of the method were evaluated.The results showed that HPLC method could accurately and precisely analyze the content of DOX in liposomes in vitro,and the liposome materials in liposomes had no obvious interference to the detection.The method meets the requirements of doxorubicin in vitro quantitative analysis.UPLC/MS/MS analysis method can accurately and precisely analyze the content of DOX in brain tissue,and the analysis method can eliminate the interference of biological matrix in brain tissue on DOX detection.The UPLC/MS/MS analytical methodology meets the requirements of DOX in vivo quantitative analysis.2.Prescription optimization of CPPs modified liposome membrane:ethanol injection method combined with the ammonium sulfate gradient method active drug preparation of CPPs modified DOX liposomes（R8PLP）,using particle size,the encapsulation efficiency as evaluation indexes to optimize ratio of cationic lipids/OA-R8/ePC/cholesterol/DSPE-PEG2000 in R8PLP,kinds of cationic phospholipids,concentration of ammonium sulfate and volume ratio of lipid phase/ammonium sulfate solution.The best conditions for the preparation of R8PLP are DOTAP as cationic phospholipids,DOTAP/OA-R8/ePC/Chol/DSPE-PEG=20/25/18/32/3,aliphatic/aqueous phase=1/10,ammonium sulfate concentration of250μM,stirring speed of 300 rpm,injection rate of 5.5 mL/min to prepare R8PLP.DOX active drug loading condition:the R8PLP drug loading was completed with a lipid ratio of 1:10,a pH value of 7.4 and a temperature of 45℃for 30 mins.3.Characterization of liposomes modified by CPPs:firstly,OA-R8 modified liposomes used for DOX delivery to the brain were prepared,and then the physicochemical properties of the liposomes were characterized and also evaluated the anti-tumor activity and cell uptake of liposomes in vitro.The surface morphology of R8PLP was spherical,with smooth surface,good dispersibility and uniform particle size of about 100 nm.R8PLP of zeta potential of+12.1 mV;R8PLP was stable in 10.0%serum,PBS or deionized water at 37℃and 4℃for 48 h.The lyophilized R8PLP was stable in 10.0%serum,PBS or deionized water at 37℃and 4℃for 48 h.DOX,LP,PLP,R8LP and R8PL showed significant anti-tumor activity in U87 and GL261 cells,and the anti-tumor activity was time dependent.After 24 h of administration,the activity of OA-R8 modified liposomes（R8LP,R8PL）was significantly higher than that of adriamycin,LP and PLP,suggesting that OA-R8 modified liposomes could improve the anti-tumor activity of DOX in U87 and GL261cells.Finally,the blood-brain barrier passability of OA-R8 modified liposomes（R8PLP）was evaluated:compared with PLP,R8PLP had a significant accumulation effect in brain tissue,and the AUC_0.5-12h area under the curve when R8PLP was treated in brain was 2.4 times that of PLP.However,the half-life of R8PLP(t_1/2)was similar to that of PLP（3.47 vs 3.94 h）,suggesting that OA-R8 modified liposomes had better BBB penetration and brain tissue accumulation.4.CPPs and Tf modified liposomes for the targeted treatment of glioma:this chapter on the basis of preparation of Tf-R8PLP,discussed the Tf content on the liposome targeting property and anti-tumor effect,the preparation of CPPs and Tf were modified（Tf-R8PLP）,and particle size of liposomes,stability in the serum,encapsulation efficiency and evaluate the drug release in vitro performance.In addition,in vitro cell uptake of Tf-R8PLP was analyzed.The glioma model of nude mice was established,and the in vivo anti-tumor efficacy of Tf-R8PLP was evaluated.After Tf-R8PLP treatment,the distribution of the drug in tissues in nude mice and the pathology of organs in nude mice including brain,heart,heart,liver,spleen,lung and kidney were evaluated.The best preparation condition of Tf-R8PLP was 1/100 mole ratio of R8PLP/Tf-DSPE-PEG.Tf-R8PLP particle size of 128.64 nm,ζ-potential was+6.81 mV,Tf concentration is 16.15μg/mL;Tf-R8PLP has spherical structure and uniform particle size,and the stability of Tf-R8PLP in serum is good.Both R8PLP and Tf-R8PLP showed controlled release behavior.Tf-R8PLP had the highest tumor growth inhibition rate,and the in vivo anti-tumor activity of Tf-R8PLP was significantly higher than that of R8PLP and DOX.The survival time of mice in Tf-R8PLP group（25 days）was longer than that in normal saline group（20 days）,DOX group（22 days）and R8PLP group（24 days）,and Tf-R8PLP had significant effect on the treatment of glioma.更多还原