乳铁蛋白修饰白藜芦醇纳米粒的制备及其脑靶向作用的初步研究

发布时间：2018-04-17 16:59

  本文选题：乳铁蛋白 + 白藜芦醇　； 参考：《遵义医学院》2017年硕士论文

【摘要】：目的:制备乳铁蛋白修饰的白藜芦醇纳米粒(Lactoferrin modified Resveratrol Nano Particles,Lf-Res-NPs),考察其在小鼠体内药物代谢动力学,观察其对阿尔茨海默病(Alzheimer’s disease,AD)模型大鼠的干预作用,初步评价其脑靶向作用。方法:(1)界面聚合法制备白藜芦醇纳米粒(Resveratrol Nano Particles,Res-NPs),星点设计优化处方和工艺,采用羟基聚乙二醇马来酰亚胺(Hydroxyl-PEG-Maleimide,OH-PEG-MAL)的-OH基团链接Res-NPs,MAL基团与活化后的乳铁蛋白巯基结合,制备Lf-Res-NPs,激光粒度分析仪检测其粒径,透射电镜观察其形态。(2)SPF级昆明小鼠270只,雄性,随机分为3组:Res溶液组、Res-NPs组和Lf-Res-NPs组。常规饲养1周后腹腔注射给药,给药后0.17、0.5、0.75、1、2、6、10、12、24h采血,每个时间点10只小鼠。采用HPLC法分析血浆及组织中药物浓度,计算药代动力学参数和各组织靶向指数。(3)SPF级SD大鼠50只,雌雄各半,随机分为正常组、AD模型组、Res溶液组、Res-NPs组和Lf-Res-NPs组,每组10只。正常组每天腹腔注射生理盐水和灌胃蒸馏水,其余各组腹腔注射D-半乳糖150mg·kg-1·d-1和灌胃三氯化铝10mg·kg-1·d-1,连续65d。第31d正常组和AD模型组腹腔注射生理盐水,药物干预组腹腔注射不同剂型Res,Res给药剂量为30mg·kg-1·d-1。造模65d后,Morris水迷宫检测大鼠认知功能;比色法检测血清及脑组织中谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-PX)、超氧化物歧化酶(Superoxide dismutase,SOD)活力及丙二醛(Malondialdehyde,MDA)含量;免疫组织化学染色观察海马β-淀粉样蛋白1-42(βeta amyloid peptides1-42,Aβ1-42)及半胱氨酸天冬氨酸蛋白酶3(Cysteinecontaining aspartate-specific protease3,Casepase-3)表达;ELISA法检测海马组织Aβ1-42的表达;Western Bolt检测海马β-淀粉样前体蛋白(βeta-Amyloid precursor protein,APP)、Caspase-3及核转录因子-κB(Nuclear factor-κB,NF-κB)蛋白的表达。结果:(1)(1)Res-NPs制备最佳处方:油水体积比4.0m L:8.0m L,水相p H 2.0,右旋糖酐-70 50mg,Res 48mg,BCA 52μL。其粒径(229.02±4.30)nm,载药量(10.13±0.96)%,包封率(80.36±2.70)%;(2)在p H7.4 PBS释放介质中,Res溶液2h累积释放Res(80.26±1.61)%,8h累积释放Res(86.16±0.10)%;Res-NPs 2h累积释放Res(56.77±2.47)%,8h累积释放Res(73.11±7.14)%,72h累积释放Res(80.24±2.78)%;Res-NPs中加入血浆时,2h累积释放Res(56.62±0.30)%,8h时累积释放Res(82.22±3.22)%,72h累积释放Res(87.96±0.72)%;(3)Lf-Res-NPs粒径(240±4.652)nm,透射电镜鉴定Lf与Res-NPs成功连接。(2)(1)药代动力学结果显示:与Res溶液组药时曲线下面积(AUC(0-∞))、生物半衰期(t1/2z)、达峰时间(Tmax)、达峰浓度(Cmax)和清除率(CLz/F)比较,Res-NPs组和Lf-Res-NPs组AUC(0-∞)、t1/2z、Tmax、Cmax增加,CLz/F降低(P0.05);与Res-NPs组比较,AUC(0-∞)、t1/2z、Tmax、Cmax增加,CLz/F降低(P0.05);(2)Res溶液组和Res-NPs组药物主要集中在肝和肾组织,Lf-Res-NPs组药物主要集中在肝、脾和脑组织,给药120min后Res溶液组脑靶向指数最大值为14.263%,Res-NPs组脑靶向指数最大值为20.980%,Lf-Res-NPs组脑靶向指数最大值为26.879%,其Lf-Res-NPs组比Res-NPs组增加1.3倍,比Res溶液组增加1.9倍;且给药后10、12、24h Lf-Res-NPs组脑靶向指数显著高于其余两组,差异有统计学意义(P0.05)。(3)(1)水迷宫定位航行结果显示:与正常组比较,AD模型组1-4d逃避潜伏期延长(P0.05);与AD模型组比较,不同剂型Res干预的各组2-4d逃避潜伏期缩短(P0.05);与Res溶液组比较,Res-NPs组和Lf-ResNPs组在第3-4d逃避潜伏期缩短(P0.05);与Res-NPs组比较,Lf-Res-NPs组在第4d逃避潜伏期缩短(P0.05);空间探索结果显示:与正常组比较,AD模型组穿环次数和滞留时间减少(P0.05);与AD模型组比较,不同剂型Res干预的各组穿环次数与滞留时间增加(P0.05);不同剂型Res组间比较,Lf-Res-NPs组穿环次数与滞留时间增加(P0.05)。(2)与正常组比较,AD模型组血清及脑组织内SOD和GXH-PX活力降低,MDA含量升高(P0.05);与AD模型组比较,Res-NPs组和Lf-Res-NPs组血清及脑组织SOD和GSH-PX活力升高,MDA含量降低(P0.05);不同剂型Res组间比较,Lf-Res-NPs组血清及脑组织SOD和GSH-PX活力增加,MDA含量降低(P0.05)。(3)免疫组化结果显示:与正常组比较,AD模型组大鼠海马Casepase-3和Aβ1-42蛋白表达增加(P0.05);与AD模型组比较,不同剂型Res干预的各组大鼠海马Casepase-3和Aβ1-42蛋白表达降低(P0.05);不同剂型Res组间比较,Lf-Res-NPs组大鼠海马Casepase-3和Aβ1-42蛋白表达降低(P0.05)。(4)ELISA检测结果显示:与正常组比较,AD模型组大鼠海马Aβ1-42蛋白表达增加(P0.05);与AD模型组比较,不同剂型Res干预的各组大鼠海马Aβ1-42蛋白表达降低(P0.05);不同剂型Res组间比较,Lf-Res-NPs组大鼠海马Aβ1-42蛋白表达降低(P0.05)。(5)Western bolt结果显示:与正常组比较,AD模型组大鼠海马Casepase-3、NF-κB和APP蛋白表达增加(P0.05);与AD模型组比较,不同剂型Res干预的各组大鼠海马Casepase-3、NF-κB、APP蛋白表达降低(P0.05);不同剂型Res组间比较,Lf-Res-NPs组大鼠海马Casepase-3、NF-κB、APP蛋白表达降低(P0.05)。结论:(1)制备得到粒径为(240±4.652)nm Lf-Res-NPs。(2)Lf-Res-NPs可延长Res在小鼠体内的作用时间,增加Res在小鼠体内的生物利用度,提高Res的脑靶向作用,减少其他组织对Res的摄取。(3)Lf-Res-NPs可递送更多Res进入脑内,增加脑内Res浓度,提高抗氧化能力,抑制海马神经元APP和Aβ1-42蛋白表达,减轻神经元凋亡和炎症反应,效果较Res溶液和Res-NPs好。
[Abstract]:Objective: Resveratrol nanoparticles modified lactoferrin (Lactoferrin modified Resveratrol Nano Particles, Lf-Res-NPs), to investigate the in vivo drug metabolism in mice to observe the dynamics of Alzheimer's disease (Alzheimer 's disease, AD) intervention model rats, evaluate its brain targeting effect. Methods: (1) preparation of resveratrol nanoparticles by interfacial polymerization (Resveratrol Nano, Particles, Res-NPs), optimized design, using hydroxyl polyethylene glycol maleimide (Hydroxyl-PEG-Maleimide, OH-PEG-MAL) -OH groups link Res-NPs, MAL group combined with activated lactoferrin thiol, preparation of Lf-Res-NPs, laser particle size analyzer and particle size, were observed morphology of TEM. (2) SPF 270 Kunming mice, male, were randomly divided into 3 groups: Res solution group, Res-NPs group and Lf-Res-NPs group. After 1 weeks of feeding abdominal routine Injection after administration of blood 0.17,0.5,0.75,1,2,6,10,12,24h, 10 rats in each time point were analyzed by HPLC assay. The drug concentration in plasma and tissues, pharmacokinetic parameters and the target to calculate the index. (3) SPF 50 SD rats, male and female, were randomly divided into normal group, AD model group. The solution of Res group, Res-NPs group and Lf-Res-NPs group, 10 rats in each group. The normal group received intraperitoneal injection of saline and distilled water, the other groups were intraperitoneally injected with D- galactose 150mg - kg-1 - D-1 and 10mg - kg-1 - alchlor intragastric administration of D-1, the continuous 65d. 31d normal group and AD model group by intraperitoneal injection of saline. The drug intervention group by intraperitoneal injection of different dosage of Res, Res dosage was 30mg - kg-1 - d-1. model 65D, water maze Morris; ratio of serum and brain tissue glutathione peroxidase assay enzyme (Glutathione peroxidase, GSH-PX), super Superoxide dismutase (Superoxide dismutase, SOD) activity and malondialdehyde (Malondialdehyde, MDA) content of hippocampus were observed; amyloid beta protein 1-42 immunohistochemical staining (ETA amyloid beta peptides1-42, A beta 1-42) and caspase 3 (Cysteinecontaining aspartate-specific, protease3, Casepase-3) to detect the expression of A beta expression; hippocampus ELISA 1-42; Western Bolt was detected in the beta amyloid precursor protein (beta eta-Amyloid precursor protein, APP, Caspase-3) and nuclear factor kappa B (factor- K Nuclear B NF- K B) protein expression. Results: (1) Res-NPs (1) prepared by the best prescription: the volume ratio of oil to water 4.0m L:8.0m L, aqueous P H 2, -70 50mg Res 48mg, dextran, BCA 52 L. diameter (229.02 + 4.30) nm, drug loading (10.13 + 0.96)%, the encapsulation rate (80.36 + 2.70)%; (2) in the P H7.4 PBS release medium, Res solution of 2H accumulation the release of Res (80. 26卤1.61)%,8h绱�绉�閲婃斁Res(86.16卤0.10)%;Res-NPs 2h绱�绉�閲婃斁Res(56.77卤2.47)%,8h绱�绉�閲婃斁Res(73.11卤7.14)%,72h绱�绉�閲婃斁Res(80.24卤2.78)%;Res-NPs涓�鍔犲叆琛�娴嗘椂,2h绱�绉�閲婃斁Res(56.62卤0.30)%,8h鏃剁疮绉�閲婃斁Res(82.22卤3.22)%,72h绱�绉�閲婃斁Res(87.96卤0.72)%;(3)Lf-Res-NPs绮掑緞(240卤4.652)nm,閫忓皠鐢甸暅閴村畾Lf涓嶳es-NPs鎴愬姛杩炴帴.(2)(1)鑽�浠ｅ姩鍔涘�︾粨鏋滄樉绀，

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