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类型 基础研究 预答辩日期 2018-05-16
开始(开题)日期 2016-10-28 论文结束日期 2018-03-09
地点 江苏省生物材料与器件重点实验室 论文选题来源 其他项目    论文字数 5.8 (万字)
题目 肺部给药的西那普肽微泡制剂研究
主题词 肺部给药,西那普肽,微气泡,肺表面活性物质,急性肺损伤
摘要 肺部表面活性物质是II型肺泡上皮细胞合成和分泌的一种磷脂-蛋白复合物, 它具有降低肺泡表面张力、维持肺泡稳定性、增强肺免疫功能、减轻肺部炎症反应等多种功能,是维持正常呼吸功能的重要物质。目前,国内外已有多种动物来源的肺表面活性物质用于肺损伤类疾病的治疗,但天然来源的药品存在批次间质量不均一,同时存在动物源性产品传播动物相关疾病风险性的问题,而合成肺表面活性物质药物能够规避该类问题。Surfaxin?是目前唯一上市的全合成肺表面活性药物,虽然在呼吸窘迫综合征治疗领域具有广泛的应用,但作为普通的混悬剂剂型,仍有许多不足之处,例如单次给药剂量大,治疗方法单一,生物利用度有待于进一步提高等。因此,该类药物的剂型及治疗方式有待进一步开发。 为了采用Surfaxin?进行相关药物新剂型的研发,本论文系统地研究了Surfaxin?混悬剂的制备工艺并建立其含量检测方法。同时,基于Surfaxin?处方组分构建了一种肺部给药用新型微泡类药物制剂,该制剂形式以磷脂类成分为膜壳,对Surfaxin?处方中主要活性成分西那普肽进行包载,较全面的评价了该制剂的药效学性能,着重研究了多肽对于微泡的稳定性机制及微泡结合超声干预对炎症肺泡上皮细胞功能及肺损伤动物模型的影响。具体内容包括以下几个部分: (1)采用分散法制备工艺,以外观性状、沉降容积比、再分散性及含量为指标,通过对加液研磨法和高速剪切法两种不同制备工艺所得西那普肽混悬剂质量稳定性的考察,确定西那普肽混悬剂最优制备工艺。采用高效液相色谱法(HPLC)对西那普肽混悬剂的含量测定方法进行研究,建立西那普肽HPLC含量检测标准:色谱柱为Diamonsil C18(250 × 4.60 nm,5 μm),流动相为0.1 %三氟乙酸(TFA)的乙腈及0.1% TFA的水,梯度洗脱乙腈:水=40 : 60,25 min后改为水:乙腈=90 : 10,进样量为20 μL,流速1 mL/min,检测波长215 nm,柱温30 ℃。结果表明西那普肽浓度在0.06 mg/mL~1.5 mg/mL范围内线性关系良好(r = 0.9994),制剂样品平均回收率98.13%,RSD为1.31%。因此,实验所建立的含量测定方法可靠、灵敏、重复性好,可用于西那普肽的质量控制。 (2)基于Surfaxin?处方组分,采用超声薄膜分散法首次制备全合成肺表面活性物质微泡,并对不同浓度西那普肽微泡制剂处方进行优化筛选,确定最优处方。结果表明,载西那普肽的微气泡平均粒径及电位测定结果分别为1.82 ± 0.15 μm 和 ?55.2 ± 3.9 mV。通过荧光倒置显微镜及激光共聚焦显微镜对异硫氰酸荧光素(FITC)标记西那普肽微泡进行观察,确定了西那普肽在微泡中主要分布于膜壳层。通过透射电镜(TEM)、扫描电镜(SEM)等手段对西那普肽微泡制剂进行表征,确定西那普肽微泡的圆球形规整结构。对西那普肽脂质微泡母液进行周期3个月的稳定性实验,结果显示该处方稳定性良好,为该创新剂型进一步开发奠定基础。 (3)对不同浓度西那普肽微泡制剂的体外超声造影效果进行考察,结果显示,相较于不含西那普肽组,西那普肽组超声信号强度明显提高,且当西那普肽浓度为0.1 mg/mL时,信号强度最优,造影持续时间最长,可达到30 min。为了进一步从理论上阐明西那普肽与微气泡膜壳的相互作用,基于Martini粗粒化力场创建分子动力学模型,通过分子动力学模拟的方式阐述西那普肽微泡稳定性机理,结果发现,在一定比例范围内,西那普肽的加入能够有效降低微泡的表面张力,增加微泡的稳定性,然而当西那普肽比例升高至一定限度后,由于空间位阻效应,磷脂膜稳定性下降。 (4)最后,通过细胞实验和动物体内实验,对西那普肽微泡制剂细胞和动物水平的药效学进行评价。在西那普肽微泡制剂对损伤肺泡上皮细胞(AT Ⅱ)的相互作用中发现,与对照组相比,微泡制剂能显著改善细胞炎症因子分泌水平及SP-A表达情况,且超声干预微泡组相对于普通混悬剂组相比炎症因子分泌及SP-A表达进一步改善。进而,以肺组织病理形态、肺湿/干重比例值、IL-6、TNF-α炎症因子水平及肺表面活性蛋白表达水平为指标,建立了小鼠急性肺损伤模型,并通过微泡制剂结合超声干预治疗的方式对LPS诱导急性肺损伤动物模型进行治疗效果评价。结果显示,微泡制剂协同超声干预治疗能够显著改善动物模型组肺部水肿情况,相对于对照组,微泡超声协同组炎症因子分泌水平显著降低,SP-A分泌情况显著改善。因此,微泡制剂协同超声治疗有望为急性肺损伤类疾病治疗提供新的选择。
英文题目 The study of sinapultide microbubbles for pulmonary drug delivery
英文主题词 Pulmonary Delivery, Sinapultide, Pulmonary Surfactant, Quality Standard, Acute Lung Injury
英文摘要 Pulmonary surfactant (PS) is a complicated mixture of lipids and proteins secreted by type II alveolar epithelial cell (AT II). The main function of PS lies in maintaining normal respiratory mechanics by reducing alveolar surface tension to prevent alveolar collapse. Besides, it also plays important roles in providing uniform lung inflation, improving efficiency of airway clearance, and alleviating lung inflammatory reaction. At present, exogenous surfactant of animal origin was wildly used for the treatment of lung injury diseases, nevertheless, safety issues like the spread risk of animal related diseases remain to be improved. Besides, the ratio of surface-active proteins to lipid was uncontrollability because the quantity of the anmial-derived agents are variable from batch to batch. Therefore, the synthetic PS can be a choice to avoid such problems. Surfaxin?, the only peptide-based synthetic pulmonary surfactant in clinical application, has been approved by Food and Drug Administration (FDA) in 2012 for the treatment of neonatal respiratory distress syndrome (NRDS). As a conventional suspension formulation, the dosage form and treatment method of Surfaxin still need further exploration for large single dose as well as simple treatment. In this thesis, we systematically studied the preparation process of Surfaxin? and quality standard of sinapultide。Furthermore, we constructed a novel drug carrier of microbubbles (MBs) for pulmonary delivery based on the formulation of Surfaxin?. The shell membrane of MBs is fabricated by the phospholipid and sinapultide, which was the main components and active ingredient of Surfaxin?. The structural characterization of the MBs was evaluated systematically. Especially, the stability mechanism of the sinapultide polypeptides to MBs was studiedby simulation methods. Furthermore, the therapeutic effect of MBs integrated with ultrasound to inflammatory alveolar epithelial cell function as well as lung injury animal models were also investigated. The following points are mainly involved in the dissertation: 1. The dispersion method was used to prepare sinapultide suspension. By using sedimentation rate, redispersibility, appearance characters and quality stability of suspension as indicators, the optimal preparation process of sinapultide suspension was determined. The high performance chromotograph (HPLC) method was adopted to determine the content of sinapultide suspension, and the testing standard of sinapultide content was estabished. A Diamonsil C18 column (250×4.60 nm, 5 μm) with gradient elution system was used. The mobile phase was acetonitrile-water with 0.1% trifluoroacetic acid (TFA) was adoptedwith the initial ratio of acetonitrile to water of 40 to 60, and the ratio of 90 to 10 after 25 min. The flow rate was 1.0 mL·min-1 with wavelength of 215 nm, and the column temperature was maintained at 30 ℃. Sinapultide had a good linear relationship (r = 0.9994) with a concentration ranging from 0.06 mg/mL to 1.5 mg/mL. The average recovery as well as relative standard deviation (RSD) of preparation samples was 98.13% and 1.31%, respectively. Results showed that the establishment method to determine the content of sinapultide is reliable, sensitive and reproducible, and it could be used for the quality control of sinapultide. 2. Synthetic pulmonary surfactant microbubbles were prepared by film dispersion and ultrasonic method based on the prescription of Surfaxin?. Besides, MBs with different concentrations of sinapultide were prepared, and the optimized MBs exhibited an average diameter of 1.82 ± 0.15 μm and zeta potential of -55.2 ± 3.9 mV. A fluorescent inverted microscope was used to observe FITC-labeled sinapultide MBs, and sinapultide was found mainly existed in the membrane shell of MBs. Transmission electron microscope (TEM) and scanning electron microscope (SEM) was used to characterize the sinapultide MBs, and the spherical structure of sinapultide MBs was confirmed. Furthermore, the stability experiment demonstrated that the mother liquor to sinapultide MBs could last for three months. Therefore, the prescription had good stability for further development of innovative dosage forms. 3. The self-made agar phantom was used to investigate the contrast effect of sinapultide MBs with different concentrations in vitro. The results showed that the ultrasonic signal intensity of sinapultide MBs group was improved obviously compared with control group. When the sinapultide concentration was 0.1 mg/mL, the ultrasonicsignal intensity was the highest and could last for 30 min. Based on the experimental results, the stability mechanism of sinapultide to the MBs was studied by molecular dynamics simulation for the first time with MARTINI force field, and results showed that the addition of proper proportion of sinapultide could decrease surface tension of MBs. Accordingly, the stability of the MBs has been greatly improved. However, the stability of membrane would decrease with the concentration increase of the sinapultide due to the steric effect. 4. The sinapultide MBs on lipopolysaccharide (LPS) mediated model of alveolar epithelial cells (AT II) was studied. The results show that the sinapultide MBs could significantly improve the secretion of inflammatory cytokines and the expression of surfactant protein A (SP-A) in AT II cells. When combined with ultrasonic treatment, the secretion of inflammatory cytokines and SP-A were further improved compared with the group without ultrasound treatment. Furthermore, the in vivo experiment based on the acute lung injury (ALI) mice model was carried. The pathological morphology of lung tissue, the ratio of lung wet/ dry weight, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and SP-A expression level were used as indexes. Results showed that the treatment of MBs combined with ultrasound could significantly improve pulmonary edema. The secretion levels of inflammatory factors as well as SP-A expression level were improved compared with the control group. Therefor, it can be concluded that microbubble combined with ultrasonic therapy maybe provide with a new option for the treatment of lung injury diseases.
学术讨论
主办单位时间地点报告人报告主题
浙江大学 2016.7.23 浙江大学干细胞与再生医学研究中心 刘东 脂质微气泡输药系统的研究
东南大学生物与医学纳米技术研究组 2015.12.02 江苏省生物材料与器件重点实验室 刘东 新生儿呼吸窘迫综合征(NRDS)治疗药物的发展及国内外现状—以SURFAXIN为例
东南大学生物与医学纳米技术研究组 2016.06.04 江苏省生物材料与器件重点实验室 刘东 肺部给药的西那普肽微泡制剂研究
东南大学生物与医学纳米技术研究组 2017.03.25 江苏省生物材料与器件重点实验室 刘东 自由气泡界面上的分子自组装
东南大学生物与医学纳米技术研究组 2017.05.08 江苏省生物材料与器件重点实验室 刘东 Surfaxin项目工作进展
东南大学生物与医学纳米技术研究组 2015.06.27 江苏省生物材料与器件重点实验室 刘东 高三尖杉酯碱长循环脂质体纳米药物的研究
中国材料研究学会 2016.9.25 南京国际青年文化中心 刘东 医学诊治一体化用智能材料
南京医科大学基础医学院 2016.10.25 南京医科大学江宁校区 刘东 脂质微纳输药系统的研究
南京大学 2017.05.21 南京金陵饭店 刘东 Sinapultide-loaded lipid microbubbles: a novel therapeutic ultrasound contrast agent
     
学术会议
会议名称时间地点本人报告本人报告题目
2016年国际华人纳米医学研讨会暨第五届全国生物与医学纳米技术博士生学术论坛 2016.6.18 苏州,中国 PEGylated liposomes of homoharringtonine for antileukemic therapy: in vitro and in vivo evaluation
The 12th European Molecular Imaging Meeting-EMIM 2017.4.6 科隆,德国 Sinapultide-loaded lipid microbubbles as an ultrasound contrast agent
     
代表作
论文名称
The smart drug delivery system and its clinical potential
Preparation and in vivo safety evaluations of antileukemic homoharringtonine-loaded liposomes
Sinapultide-loaded lipid microbubbles and the stabilization effect of sinapultide on the shells
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
孔令义 正高 教授 博导 中国药科大学
邓林红 正高 教授 博导 常州大学
吴剑卿 正高 教授 博导 南京医科大学
李玲 正高 教授 博导 东南大学
吉民 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
李艳 其他 讲师 东南大学