神经生长因子脂质体跨血脑屏障体内外研究神经生长因子脂质体跨血脑屏障体内外研究

目的研究神经生长因子（nerve growth factor,NGF）脂质体在体外血脑屏障（blood-brain barrier,BBB）模型上的通透性以及在体内脑组织的分布，并对体内外结果进行相关性分析。方法用小鼠脑微血管内皮细胞(BMVEC)建立的BBB体外实验模型，研究NGF脂质体在体外模型上的通透率；¹²⁵I-NGF和SDS-PAGE法联合使用研究NGF脂质体在脑组织的分布。结果NGF脂质体的最高包封率为34%，平均粒径小于100 nm。脂质体能够增加NGF在BBB模型上的通透率，以NGF-SSL-T为最大。脑组织药物浓度次序为NGF-SSL-T>NGF-SSL+RMP-7>NGF-SSL。体内外结果具有良好相关性。结论脂质体能够增加NGF跨越BBB的能力，RMP-7偶联在脂质体上（NGF-SSL-T）效果好于RMP-7与脂质体的简单混合（NGF-SSL+RMP-7）。     

类透明质酸壳聚糖微乳对小鼠血脑屏障通透性的影响

目的研究类透明质酸壳聚糖微乳(HAC-ME)对血脑屏障开放的促进作用。方法采用甲酰胺提取-紫外分光光度法测定小鼠各脏器中伊文思蓝(EB)的浓度，考察HAC-ME对伊文思蓝在小鼠体内组织分布的影响；荧光显微镜观察脑组织切片中伊文思蓝的荧光强度和分布。结果与未修饰的微乳比较，低浓度的HAC(<5 mg·mL^-1)表面修饰微乳可进一步促进伊文思蓝透过血脑屏障，tmax延后0.5 h左右，同时降低肝、肾组织中EB浓度。结论HAC-ME可显著增加血脑屏障的通透性，提高药物的脑趋向性，该作用与HAC的浓度有关。     

DSPE-PEG,Tween80和Brij35对伊文思蓝脂质体包封率和体内分布的影响

目的寻找与DSPE．PEG功能相似的表面活性剂，以增加脂质体的稳定性，改善其体内分布，提高靶向性。方法制备了伊文思蓝脂质体，考察了胆固醇与磷脂的比例对伊文思蓝脂质体包封率的影响；比较了用DSPE-PEG、Tween80和Brij35修饰后的伊文思蓝脂质体包封率和在大鼠体内分布状况的变化。结果伊文思蓝脂质体的包封率最高为25．30％。用DSPE-PEG、Tween80和Brij35修饰后使伊文思蓝脂质体的包封率略有下降，但差别不显著；体内分布实验结果显示：修饰后的脂质体在肝、脾和肾中伊文思蓝的浓度均有不同程度的降低，脑中伊文思蓝的浓度有所提高，而且以Tween80修饰组最显著。结论DSPE-PEG、Trween80和Brij35对伊文思蓝脂质体的包封率影响较小。Brij35对伊文思蓝脂质体的作用与DSPE-PEG相似，能提高脂质体逃避网状内皮系统吞噬的能力；Tween80主要能增加伊文思蓝脂质体在大鼠脑组织中的分布，为脑靶向脂质体的研究提供了有益信息。     

大黄酒炙前后对血脑屏障的影响

目的探讨生大黄、酒炙大黄提取液对小鼠血脑屏障通透性的影响。方法以血脑屏障示踪剂伊文思蓝（E.B.）作为判断指标,比较小鼠灌服生大黄和酒大黄提取液后脑组织的蓝染情况,并通过测定脑组织中伊文思蓝的含量来量化血脑屏障通透性改变的程度。结果与生理盐水对照组小鼠比较,受试组小鼠脑组织有明显蓝染现象,酒大黄水提液高剂量组脑组织中伊文思蓝的含量差异有显著的统计学意义（P〈0.01）。结论酒大黄水提液能够促进血脑屏障的开放。     

灯盏花素对大鼠脑创伤的保护作用

目的:观察灯盏花素对大鼠脑创伤后脑水肿、血脑屏障通透性和氧自由基的影响。方法:84只大鼠随机分为假手术组,模型组及低(25 mg/kg×2)、高(50 mg/kg×2)剂量灯盏花素组。采用液压颅脑损伤模型,脑创伤的同时尾静脉注射灯盏花素,8 h后重复给药一次。检测各组大鼠脑创伤后24 h脑组织含水量,伊文思蓝、超氧化物歧化酶(SOD)和丙二醛(MDA)的含量。结果:模型组大鼠脑创伤后脑组织含水量,伊文思蓝、MDA和SOD含量与假手术组有显著性差别。大鼠脑创伤后注射低剂量和高剂量灯盏花素均可显著降低脑组织含水量、伊文思蓝含量和MDA含量,显著增加SOD含量(P＜0．05)。结论:灯盏花素可减轻大鼠脑创伤后脑水肿,其对大鼠脑创伤的保护作用与降低血脑屏障通透性、抑制氧自由基反应有关。     

通窍活血汤对脑缺血再灌注小鼠血脑屏障通透性及脑内单胺类神经递质含量的影响

目的：观察通窍活血汤（TQHXD）对脑缺血再灌注损伤小鼠血脑屏障（BBB）通透性及脑组织中单胺类神经递质的影响。方法将昆明种小鼠随机分为假手术组,模型组,尼莫地平组,脑脉泰组,通窍活血汤低、中、高剂量组（分别为3．85,7．7,15．4 g·kg－1）。于给药第7天采用双侧颈总动脉结扎法制造急性脑缺血再灌注模型,通过测定渗出脑血管外的伊文思蓝含量分析药物对BBB通透性的影响,及采用酶联免疫法（ELISE）测定脑组织中单胺类神经递质去甲肾上腺素（NE）、多巴胺（DA）和5－羟色胺（5-HT）的含量。结果 TQHXD各剂量组均能降低小鼠脑中伊文思蓝的含量,且均能阻止脑损伤后单胺类神经递质的降低。结论 TQHXD对脑缺血再灌注模型小鼠有一定的保护作用。     

不同表面活性剂对伊文思蓝脂质体体内外性质的影响

目的　寻找与二硬脂酰磷脂酰乙醇胺 聚乙二醇 (DSPE PEG)功能相似的表面活性剂 ,以增加脂质体的稳定性 ,改善其体内分布 ,提高靶向性。方法　制备伊文思蓝 (EB)脂质体 ,考察胆固醇与磷脂的比例对伊文思蓝脂质体包封率的影响 ;比较用DSPE PEG、吐温 80 (Tween 80 )和苄泽 3 5 (Brij 3 5 )修饰后的EB脂质体的包封率和在大鼠体内组织分布状况的变化。结果　EB脂质体的包封率最高为 2 5 3 0 %。用DSPE PEG、Tween 80和Brij 3 5修饰后使EB脂质体的包封率略有下降 ,但差别不显著 ;体内分布实验结果显示 :修饰后的脂质体在肝、脾和肾中EB的浓度均有不同程度的降低 ,脑中EB的浓度有所提高 ,而且以Tween 80修饰组最显著。结论　DSPE PEG、Tween 80和Brij 3 5对EB脂质体的包封率影响较小。Brij 3 5对EB脂质体的作用与DSPE PEG相似 ,能提高脂质体逃避网状内皮系统吞噬的能力 ;Tween 80主要能增加EB脂质体在大鼠脑组织中的分布 ,为脑靶向脂质体的研究提供了有益信息     

注射用尼莫地平脂质体对大鼠全脑缺血再灌注和小鼠缺氧性损伤的保护作用

目的：研究注射用尼莫地平脂质体（NDLI）对大鼠全脑缺血再灌注和小鼠缺氧性损伤的保护作用。方法：采用小鼠常压耐缺氧实验和小鼠断头实验，观察NDLI对小鼠耐缺氧能力的影响；采用Pulsinelli四动脉结扎法略加改良制作全脑缺血模型，记录对脑电图（EEG）及翻正反射的恢复时间、脑组织匀浆伊文思蓝含量的影响。结果：NDLI能显著延长小鼠的存活时间及断头后喘气持续时间，缩短大鼠脑电图及翻正反射的恢复时间，降低脑匀浆伊文思蓝的含量。结论：NDLI对全脑缺血再灌注和缺氧性损伤有明显的保护作用。     

一甲基肼跨血脑屏障转运的动力学

目的研究一甲基肼(MMH)跨血脑屏障转运的动力学特征,为阐明其血脑屏障转运机制提供依据。方法采用原位脑灌流技术对雄性Wistar大鼠进行MMH双侧脑灌流。MMH灌流浓度分别为145,290和580mg·L-1,灌流时间为2,5,8及10min;采用对-二甲氨基苯甲醛比色法进行脑组织中的MMH浓度测定。结果MMH可以跨过血脑屏障进入脑实质,脑中MMH浓度随着灌流浓度和灌流时间的增加而呈上升趋势;而MMH在各灌流浓度下跨血脑屏障转运速度常数kin并不随灌流浓度的升高而改变,分别为(0.0240±0.0015),(0.0308±0.0041)和(0.0300±0.0041)mL·min-1·g-1。结论MMH跨血脑屏障转运属于被动扩散的膜限速模型。     

冰片对脑外伤脑血管内皮细胞iNOS表达的影响

冰片为芳香开窍药的一种，现代中医药中多用于心脑血管疾病。有研究表明冰片具有促进血脑屏障(blood brain barrier,BBB)通透性增加和引药上行的作用^[1]，另有研究发现，冰片可促进其它物质如造影剂泛影葡胺、染料伊文思蓝和药物如庆大霉素等进入脑组织中的量。为了进一步研究冰片促血脑屏障开放是否为生理性开放，以及其与病理性开放的异同，进行了下面的研究。     

The Effect of RMP-7 and its Derivative on Transporting Evens Blue Liposomes into the Brain

To investigate the effect of RMP-7 and its derivative on drug transport across blood brain barrier (BBB), RMP-7 and DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide, PEG M 3400] were conjugated under mild conditions and the reaction ratio was determined using MALDI–TOF-MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry). An endothelial cell monolayer in vitro BBB model was established and used to determine the bioactivity of RMP-7 and its derivative “opening BBB.” Horse radish peroxide (HRP), liposome (HRP-L-PEG), and Evens blue (EB) liposome (EB-L-PEG) were prepared using the reverse-phase evaporation method. HRP-L-PEG-RMP-7 and EB-L-PEG-RMP-7 were obtained by inserting DSPE-PEG-RMP-7 into the surface of liposome. The bioactivity of RMP-7 and DSPE-PEG-RMP-7 opening BBB were evaluated to determine their effect on the permeation ratio of HRP and HRP liposome across the in vitro BBB model. To evaluate the in vivo bioactivity of RMP-7 and DSPE-PEG-RMP-7 on EB transport across BBB into the brain, the indicated compounds were administered to rats. Then, brain slices were analyzed using confocal laser scanning microcopy and the EB concentration in the brain, liver, spleen, lung, and kidney was determined using the formamide–extraction–ultraviolet-spectrophotometric method. The results demonstrated that RMP-7 was conjugated with DSPE-PEG-NHS at the molecular ratio of 1:1 and the product is DSPE-PEG-RMP-7. Compared with adding HRP alone, RMP-7 and DSPE-PEG-RMP-7 improved 2- to 3-fold the transport of HRP in the in vitro BBB model. The in vivo experiments showed that DSPE-PEG-RMP-7 was better at facilitating EB transport into brain than RMP-7. The reason may be that DSPE-PEG-RMP-7 can “open BBB” as soon as the EB-L-PEG-RMP-7 reaches BBB.     

The effect of RMP-7 and its derivative on transporting Evans blue liposomes into the brain

To investigate the effect of RMP-7 and its derivative on drug transport across blood brain barrier (BBB), RMP-7 and DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide, PEG M 3400] were conjugated under mild conditions and the reaction ratio was determined using MALDI-TOF-MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry). An endothelial cell monolayer in vitro BBB model was established and used to determine the bioactivity of RMP-7 and its derivative “opening BBB.” Horse radish peroxide (HRP), liposome (HRP-L-PEG), and Evens blue (EB) liposome (EB-L-PEG) were prepared using the reverse-phase evaporation method. HRP-L-PEG-RMP-7 and EB-L-PEG-RMP-7 were obtained by inserting DSPE-PEG-RMP-7 into the surface of liposome. The bioactivity of RMP-7 and DSPE-PEG-RMP-7 opening BBB were evaluated to determine their effect on the permeation ratio of HRP and HRP liposome across the in vitro BBB model. To evaluate the in vivo bioactivity of RMP-7 and DSPE-PEG-RMP-7 on EB transport across BBB into the brain, the indicated compounds were administered to rats. Then, brain slices were analyzed using confocal laser scanning microcopy and the EB concentration in the brain, liver, spleen, lung, and kidney was determined using the formamide-extraction-ultraviolet-spectrophotometric method. The results demonstrated that RMP-7 was conjugated with DSPE-PEG-NHS at the molecular ratio of 1:1 and the product is DSPE-PEG-RMP-7. Compared with adding HRP alone, RMP-7 and DSPE-PEG-RMP-7 improved 2- to 3-fold the transport of HRP in the in vitro BBB model. The in vivo experiments showed that DSPE-PEG-RMP-7 was better at facilitating EB transport into brain than RMP-7. The reason may be that DSPE-PEG-RMP-7 can “open BBB” as soon as the EB-L-PEG-RMP-7 reaches BBB.     

The study on brain targeting of the amphotericin B liposomes

To improve transporting drugs across the Blood Brain Barrier (BBB) into the brain, RMP-7 was conjugated to the surface of liposomes containing Amphotericin B (AmB) for cerebral inflammation, because it can selectively bound to the B2 receptors on the capillary blood vessel. First, RMP-7 was conjugated to DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly (ethylenegly-col)]-hydroxy succinamide, PEG M 3400] under mild condition to obtain a predominantly 1:1 conjugate (DSPE-PEG-RMP-7), as evidenced by the Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). The second, endothelium cell was cultured on the cell insert to form an in vitro BBB model and the stereoscan microscope, electric resistance and permeation of horse-radish peroxidase (HRP) across the endothelium cell monolayer were used as indicators to evaluate the integrality of the monolayer, and then the in vitro BBB model was used to determine the bioactivity of DSPE-PEG-RMP-7 "opening" BBB. The results demonstrated the in vitro BBB model was set up, RMP-7 and DSPE-PEG-RMP-7 could improve the transporting of HRP across the BBB. The third, the liposomes containing AmB (AmB-L-PEG) was prepared by modified Film-sonication method and DSPE-PEG-RMP-7 was used to modify the AmB-L-PEG to obtain AmB-L-PEG-RMP-7. The fourth, tissue distribution of AmB in the rats of three groups was determined: Group I, AmB-L-PEG; Group II, AmB-L-PEG+RMP-7 (the physical mixture of AmB-L-PEG and RMP-7); Group III, AmB-PEG-RMP-7. The drugs were transfused into the rats through the femoral vein. The concentration of AmB in the tissue was checked using High-Performance Liquid Chromatography (HPLC) method. The rank of AmB concentration in the brain were as follows: III>II>I. The AmB concentration in the liver, spleen, lung and kidney had no significant difference. The concentration of AmB in the brain of the group III was raised several times higher than that in the other two groups, because the DSPE-PEG-RMP-7 had been inserted in the surface of AmB-L-PEG. Both the RMP-7 and AmB-L-PEG could reach BBB at the same time. When RMP-7 selectively reacted with the B2 receptor, the BBB is "opened" and AmB was transported into the brain at the same time. While in group II, the RMP-7 could improve the AmB concentration in the brain a little, because the RMP-7 and liposomes could not reach BBB at the same time. The distribution of AmB in the tissues demonstrated that the RMP-7 and its derivative had selectivity to the brain.     

The Study on Brain Targeting of the Amphotericin B Liposomes

To improve transporting drugs across the Blood Brain Barrier (BBB) into the brain, RMP-7 was conjugated to the surface of liposomes containing Amphotericin B (AmB) for cerebral inflammation, because it can selectively bound to the B2 receptors on the capillary blood vessel. First, RMP-7 was conjugated to DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly (ethylenegly-col)]-hydroxy succinamide, PEG M 3400] under mild condition to obtain a predominantly 1:1 conjugate (DSPE-PEG-RMP-7), as evidenced by the Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS). The second, endothelium cell was cultured on the cell insert to form an in vitro BBB model and the stereoscan microscope, electric resistance and permeation of horse-radish peroxidase (HRP) across the endothelium cell monolayer were used as indicators to evaluate the integrality of the monolayer, and then the in vitro BBB model was used to determine the bioactivity of DSPE-PEG-RMP-7 "opening" BBB. The results demonstrated the in vitro BBB model was set up, RMP-7 and DSPE-PEG-RMP-7 could improve the transporting of HRP across the BBB. The third, the liposomes containing AmB (AmB-L-PEG) was prepared by modified Film-sonication method and DSPE-PEG-RMP-7 was used to modify the AmB-L-PEG to obtain AmB-L-PEG-RMP-7. The fourth, tissue distribution of AmB in the rats of three groups was determined: Group I, AmB-L-PEG; Group II, AmB-L-PEG+RMP-7 (the physical mixture of AmB-L-PEG and RMP-7); Group III, AmB-PEG-RMP-7. The drugs were transfused into the rats through the femoral vein. The concentration of AmB in the tissue was checked using High-Performance Liquid Chromatography (HPLC) method. The rank of AmB concentration in the brain were as follows: III>II>I. The AmB concentration in the liver, spleen, lung and kidney had no significant difference. The concentration of AmB in the brain of the group III was raised several times higher than that in the other two groups, because the DSPE-PEG-RMP-7 had been inserted in the surface of AmB-L-PEG. Both the RMP-7 and AmB-L-PEG could reach BBB at the same time. When RMP-7 selectively reacted with the B2 receptor, the BBB is "opened" and AmB was transported into the brain at the same time. While in group II, the RMP-7 could improve the AmB concentration in the brain a little, because the RMP-7 and liposomes could not reach BBB at the same time. The distribution of AmB in the tissues demonstrated that the RMP-7 and its derivative had selectivity to the brain.     

Liposome formulated with TAT-modified cholesterol for enhancing the brain delivery

Delivery of drugs to the brain is a major challenge due to the presence of the blood-brain barrier (BBB). The cell penetrating peptide TAT, which appears to enter cells with alacrity, can pass through the BBB efficiently. With this in mind, a novel TAT-modified liposome (TAT-LIP) was developed for overcoming the ineffective delivery of normal drug formulation to the brain. Targeting liposomal formulations are always composed of modified phospholipids as an anchor. However, cholesterol, another liposomal component, which was more stable and cheaper, has not been fully investigated as an alternative anchor. In this study, TAT was covalently conjugated with the cholesterol to prepare the liposome. The cellular uptake by brain capillary endothelial cells (BCECs) of rats and the mechanism of TAT-LIP pathway of endocytosis was explored. The blood brain barrier model in vitro was established to evaluate the transendothelial ability crossing the BBB and its transport mechanism. The biodistribution of each formulation was further identified. The results showed that the positive charge of the TAT-LIP played an important role in enhancing its brain delivery. The absorptive endocytosis might be one of the mechanisms of TAT-LIP crossing the BBB. In conclusion, the experimental data in vitro and in vivo indicated that the TAT-LIP was a promising brain drug delivery system due to its high delivery efficiency across the BBB.     

Gene delivery to brain cells with apoprotein E derived peptide conjugated to polylysine (apoEdp-PLL)

A promising strategy to carry genetic material to brain cells either in vitro or in vivo is using the LDL receptor (LDLr) on blood-brain barrier. LDLr naturally help to low density lipoproteins (LDL(S)) transporting across the BBB by endocytosis. Here we present the idea of using the LDLr-mediated pathway for transporting genetic material to brain cells. A tandem dimer Sequence of apoprotein-E (apoE) (141-150) conjugated to polylysine sequence was used as a novel DNA Delivery vector for transfecting of brain cells either in vitro or in vivo. DNA condensation occurs with this vector because electrostatic interaction between DNA and polylysine. The vector favors to protection of DNA from enzymatic degradation and also helps to DNA carrying in blood stream to reach BBB and transport it to brain cells and eventually help DNA expression in target cells. These results suggest a novel gene delivery vector for gene therapy of brain disease.     

RMP-7对两性霉素B脂质体跨血脑屏障能力的药效学研究RMP-7对两性霉素B脂质体跨血脑屏障能力的药效学研究

目的研究两性霉素B脑靶向脂质体对小鼠脑膜炎的治疗作用。方法用薄膜超声法制备两性霉素B脂质体和两性霉素B脑靶向脂质体,测定其包封率和浓度;建立小鼠的脑膜炎模型,研究了两性霉素B脂质体的疗效。结果两性霉素B脂质体的包封率为(93.3±1.8)%;脑内注射白色念珠球菌2 h使小鼠形成脑膜炎模型;将两性霉素B制成脑靶向脂质体,能显著延长脑膜炎小鼠的生存期。结论与普通两性霉素B脂质体相比,两性霉素B脑靶向脂质体能显著提高对小鼠脑膜炎的治疗作用,使小鼠的存活期延长一倍。     

Neuroprotection Against Degeneration of SK-N-MC Cells Using Neuron Growth Factor-Encapsulated Liposomes with Surface Cereport and Transferrin

Liposomes with Cereport (RMP-7) and transferrin (Tf) (RMP-7/Tf/liposomes) were employed to target the blood–brain barrier (BBB) and to inhibit the degeneration of neurons insulted with fibrillar β-amyloid peptide 1–42 (Aβ_1–42). Neuron growth factor (NGF)-encapsulated RMP-7/Tf/liposomes (RMP-7/Tf/NGF-liposomes) were used to permeate a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes (HAs) and to treat Aβ_1–42-attacked SK-N-MC cells. An increase in RMT-7 concentration increased the particle size, zeta potential, propidium iodide (PI) permeability, and NGF permeability, but decreased the cross-linking efficiency of RMT-7, viability of HBMECs and HAs, and transendothelial electrical resistance (TEER). In addition, an increase in Tf concentration enhanced the particle size, viability of HBMECs, HAs, and SK-N-MC cells, PI permeability, and NGF permeability, but reduced the zeta potential, cross-linking efficiency of RMT-7 and Tf, and TEER. RMP-7/Tf/NGF-liposomes can transport NGF across the BBB and improve the neuroprotection for Alzheimer's disease therapy in preclinical trials. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2484–2497, 2014