【作者】 周文英；

【导师】 金义光； 杜丽娜；

【作者基本信息】 河南大学 ， 药剂学， 2009， 硕士

【摘要】 为解决微米级超声造影剂只能进行肿瘤血管内成像或治疗及现存纳米级超声造影剂成像效果差、不能治疗的问题,本课题在微泡及纳米泡超声造影剂研究基础上,进行了肿瘤靶向超声敏感纳米泡的研究。首先进行成膜材料聚乳酸-羟基乙酸聚乙二醇单甲醚共聚物（PLGA-PEG）的合成,并对其性质进行考察。然后以抗肿瘤药阿霉素（DOX）为模型药物,具有两亲性的PLGA-PEG为成膜材料,具有气液相变性质（相变温度29℃）全氟戊烷（PFP）为成像气体,制备得到载DOX纳米泡,并对其体内外行为进行考察,具体研究内容如下:1.成膜材料的合成以生物相容性好、可生物降解的聚乳酸-羟基乙酸（PLGA）为疏水段,聚乙二醇单甲醚（mPEG）为亲水段,通过选择合适的反应条件及分离纯化条件合成得到两嵌段共聚物PLGA-PEG,并对其结构进行确证。2.成膜材料的性质对PLGA-PEG的溶解性能进行了测定,其不溶于水及醇类溶剂如甲醇和乙醇等,易溶于卤代烷烃如氯仿和二氯甲烷等,溶于四氢呋喃、乙酸乙酯和丙酮。用Langmuir膜天平对PLGA-PEG单分子膜性质进行了考察,PLGA-PEG有很好的形成单分子膜的能力,表明其有很好的两亲性。DOX（一定量三乙胺碱化）在pH=7.4 PBS表面能很好的形成单分子膜,DOX可插入到PLGA-PEG的单分子膜中,DOX与PLGA-PEG混合后形成的单分子膜有很好的静动态稳定性。3.DOX胶束的制备及结构特点建立了HPLC体外分析方法,以胶束粒径分布、溶液外观、载药量、包封率等为评价指标,进行了DOX胶束的制备方法选择及处方工艺优化,用低温丙酮注入法制备得到了粒径分布均匀、载药量、包封率均较高的DOX胶束,工艺重现性好。负染法透射电镜观察其结构为球形的以疏水区为核、亲水区为壳的核—壳结构,平均粒径为61 nm左右。4.DOX纳米泡的制备及结构特点以纳米泡粒径分布、粒径随温度变化为评价指标,进行DOX纳米泡制备方法的选择及处方工艺优化,用超声注入法制备得到粒径均匀的DOX纳米泡（室温下以纳米乳形式存在）,工艺重现性好,激光粒度分析仪测其平均粒径为168nm,光学显微镜观其50℃孵育后纳米泡融合形成外观为圆形表面较红的微泡,荧光显微镜观其结构为中空的壳—核结构,DOX载在壳壁的疏水区。5.DOX纳米泡相关性质考察以粒径为评价指标对DOX的温度敏感性、超声敏感性、超声合并性、超声释药性、稀释稳定性、放置稳定性等进行考察,结果表明DOX纳米泡的粒径能随温度变化而变化,纳米泡在超声下可合并成微泡,微泡在继续超声时可破裂快速释放药物,且在PH=6.5 PBS中比PH=7.4 PBS中更易释放药物。纳米泡在4℃保存短期内稳定性较好,稀释50倍后粒径不变,稀释后在37℃pH=7.4 PBS中孵育15min DOX仅释放0.79%,稳定性很好。6.DOX纳米泡在动物体内的药代动力学和组织分布建立了小鼠右侧腋窝皮下接种H₂₂肝癌模型,建立了准确、快速、灵敏的HPLC体内分析方法。对DOX纳米泡静脉注射后在正常鼠与荷瘤鼠体内药代动力学与组织分布分别进行了研究。DOX纳米泡在荷瘤鼠体内半衰期(t_1/2)要比正常鼠明显短,荷瘤鼠t_1/2为570±148min,而正常鼠t_1/2为1019±655min。DOX纳米泡在正常鼠肝脏中分布最多（2.77%）,荷瘤鼠肿瘤部位分布最多（1.11%）,在正常鼠与荷瘤鼠其余组织中均有少量分布（0～0.27%）,结合药动数据,证明DOX纳米泡有很好的肿瘤靶向性。7.DOX纳米泡荷瘤鼠药效学建立了小鼠右侧腋窝皮下接种H₂₂肝癌模型,对DOX纳米泡静脉注射后荷瘤小鼠药效学进行了研究,实验组与对照组间瘤重有显著性差异（P＜0.01）,药效显著,超声组与未超声组间也有显著性差异（P＜0.01）,超声更有助于DOX发挥药效。证明DOX纳米泡有被动靶向性且在超声下能快速释药进入肿瘤细胞内发挥药效。本文制备得到了粒径分布均匀,低温保存短期内稳定性好的肿瘤靶向超声敏感的纳米泡,证明了纳米泡的壳—核结构,DOX载在泡壁上,在荷瘤鼠体内有很好的肿瘤靶向性,且在超声下能更好地发挥药效。更多还原

【Abstract】 Microbubble ultrasound contrast agent （UCA）can only image and exert therapy within tumor vessel and there existed many problems of nanoscale ultrasound contrast agent such as poor imaging and can’t therapy. To solve the problem mentioned above, in the base of microbubble and nanobubble UCA, tumor-targeted nanobubble which was sensitive to ultrasound was investigated in this paper.First, the amphipathic polymer, poly （D,L-lactide-co-glycolide） -block-monomethoxy poly（ethylene glycol） （PLGA-PEG） was synthesized and its properties were also investigated. Second, tumor targeted nanobubble was prepared with doxorubicin （DOX） as the model drug, PLGA-PEG as film-former, perfluoropentane（PFP）（boiling point 29℃）as the imaging gas. Then its behavior in vitro and in vivo was investigated The main contents were described in details as follows:1. Synthesis of film-former. The diblock PLGA-PEG was synthesized using poly（D,L-lactide-co-glycolide） （PLGA） as a hydrophobic segment and monomethoxy poly（ethylene glycol） （mPEG） as a hydrophilic segment. By selecting appropriate reaction and purification conditions, PLGA-PEG was obtained and purified.2. Characteristics of film-former. The solubility of PLGA-PEG was determined. PLGA-PEG can dissolve easily in alkylogen, such as dichlormethane（DCM）and chloroform, and dissolve in tetrahydrofuran（THF）, acetone, acetoacetate. But it can’t dissolve in water and alcohol sovent, such as methanol and ethanol. Monomolecular film properties of PLGA-PEG was determined by Langmuir balance. The results showed that PLGA-PEG had good ability to form monomolecular film and thus certificated that it was amphiphilic. DOX（alkalinized by triethylamine）can form monomolecular film in pH=7.4 PBS and DOX can insert into the monomolecular film of PLGA-PEG. The mixing monomolecular film of DOX and PLGA-PEG had good static and dynamic stability.3. Preparation and Characterization of DOX micelles. HPLC analysis method in vitro of DOX was established. The formulation and preparation methods of DOX micelles were optimized with the particle size distribution, loading amount and encapsulation efficiency as the evaluation index. Finally, the DOX micelles were prepared by acetone injection method at low tempture. The size distribution of DOX micelles obtained was uniform, loading amount and encapsulation efficiency high. The technique reproducibility was very good. Negative staining TEM imaging showed the structure of micelles was globular and core-shell, with hydrophobic domain forming core and hydrophilic domain forming shell. The average size was 61nm.4. Preparation and Characterization of DOX nanobubbles. The preparation methods and technique of DOX nanobubbles were optimized with the size distribution, size change with temperature as the evaluation index. The DOX nanobubbles were prepared by the ultrasound injection method. Its size distribution was uniform, the technique reproducibility good, average size 168nm. After incubated in 50℃water, the structure was observed by light microscope, and the results showed that it was round with red surface. And the fluorescence microscope showed it was core-shell and DOX was localized in the outward hydrophobic compartment.5. Characteristics of DOX nanobubbles. The temperature sensibility, ultrasound sensibility, ultrasound incorporation, ultrasound-induced releasing drug, diluting stability was investigated with the size change as the evaluation index. The results showed that the size of DOX nanobubbles changed with the temperature. Ultrasound could make nanobubbles incorporate into microbubbles, and microbubbles disrupted when ultrasound power was increased, thus releasing DOX rapidly, more easily in pH=6.5 PBS. Nanobubble was stable in short term when stored in 4℃, and could stand dilution of 50 times. Nanobubble was incubated in 37℃water for 15 min after diluted and only 0.79% DOX was released. The stability was very good.6. Pharmacokinetics and tissue distribution of DOX nanobubbles. Subcutaneous tumors model was established using Kunming mice. HPLC analysis method in vivo was established. Pharmacokinetics and tissue distribution of DOX nanobubbles in normal and tumor-bearing mice were investigated, respectively. The elimination t_1/2 in normal and tumor-bearing mice was 1019±655 min and 570±148 min, respectively. DOX nanobubbles were distributed in heart, liver, spleen, lung and kidney in two model animals after iv 0.5h, some in heart,spleen, lung and kidney（0～0.27%）, the most in liver（2.77%）of normal mice and tumor（1.11%）of tumor-bearing mice, respectively. Combined with pharmacokinetics results, it demonstrated that DOX nanobubbles had good tumor targeting.7. Pharmacodynamics of DOX nanobubbles. Subcutaneous tumors model was established using Kunming mice. Pharmacodynamics of DOX nanobubbles were investigated using tumor-bearing mice. The tumor weight differentiated significantly between the control and experimental group（p<0.01）. There were distinguished differences between the ultrasound and non-ultrasound group （p<0.01）. The results showed that DOX nanobubbles had good passive targeting action and ultrasound can facilitate it into tumor cells.Tumor targeting and ultrasound-sensible DOX nanobubbles with uniform size distribution and good stability when stored in 4℃were prepared in this paper. Its core-shell structure was demonstrated and DOX was localized in the hydrophobic wall of the nanobubbles. It had good tumor targeting for tumor- bearing mice after iv, and also exerted good pharmacodynamic action when using ultrasound.更多还原