磁导向纳米磁性药物载体对迟发型脑血管痉挛干预的实验研究

汪子文 桂铮 卢学刚 郭栋 朱明 李臻 韩新巍 于耀宇

(1郑州大学第一附属医院介入科，河南 郑州 450001; 2西安交通大学理学院材料物理系，陕西 西安 710049)

·脑血管疾病诊治研究·

磁导向纳米磁性药物载体对迟发型脑血管痉挛干预的实验研究

汪子文1桂铮1卢学刚2郭栋1朱明1李臻1韩新巍1于耀宇1*

(1郑州大学第一附属医院介入科，河南 郑州 450001;2西安交通大学理学院材料物理系，陕西 西安 710049)

目的探讨在磁导向下纳米磁性药物载体(MNA-DPD)对迟发型脑血管痉挛(DCVS)干预的有效性。方法健康成年新西兰白兔100只，随机分为5组：正常组(n=20)，处理组(n=20)：溶剂对照组(DMSO)、纳米磁性蛋白组(MNA)、2-2'联吡啶组(DPD)和纳米磁性药物载体组(MNA-DPD)。处理组采用二次注血法制作DCVS模型，然后每天分别通过腰大池给予0.01% DMSO、MNA、DPD和MNA-DPD，剂量50 mg/kg。MNA组、MNA-DPD组给予外加磁场磁导向。在给药7 d时处死动物，取其基底动脉行HE染色并测定管径，同时行普鲁士蓝染色。Western Blot定量分析给药后7 d动物模型基底动脉增殖细胞核抗原(PCNA)、血管内皮生长因子(VEGF)、c-Myc蛋白和p53蛋白表达水平。结果HE染色和普鲁士蓝染色显示DMSO组、MNA组基底动脉明显痉挛，DPD组基底动脉可见痉挛，MNA-DPD组基底动脉基本正常的形态(Plt;0.05)。并可见蛛网膜下腔的纳米磁性微粒被染成蓝色。Western Blot定量分析PCNA、p53、c-Myc、VEGF蛋白在DMSO组、MNA组和DPD组呈现高表达，MNA-DPD组呈现低表达(Plt;0.05)。结论在磁导向下，腰大池给予纳米磁性药物载体，能够有效地抑制DCVS。

磁导向纳米磁性药物载体; 迟发性脑血管痉挛; 蛛网膜下腔出血

自发性蛛网膜下腔出血(subarachnoid hemorrhage, SAH)85%以上是脑动脉瘤破裂所致，迟发性脑血管痉挛(delayed cerebral vasospasm, DCVS)是SAH后病情加重，致残、致死的主要原因。约70%以上SAH出现DCVS[1-2]。目前流行的DCVS治疗是应用Ca2+拮抗剂，疗效不理想，至今仍存在争议。本研究根据DCVS相对肯定的病理机制，经过DCVS模型腰大池途径，利用磁导向将纳米磁性药物载体靶向聚集于DCVS部位缓释药物，增加药物的有效浓度。有效改善并阻止DCVS病理进程。

材料与方法

一、实验动物

健康成年新西兰大白兔100只 (郑州大学实验动物中心提供), 雌雄不限，平均体重2.5 kg。

二、实验试剂和仪器

兔抗抑癌基因p53抗体、兔抗血管内皮生长因子(vascular endothelial growth factor, VEGF)抗体、小鼠抗增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)抗体和兔抗原癌基c-myc抗体(Chemicon, Temecula, CA, USA)；苏木素伊红染色试剂盒(中国碧云天)；纳米磁性药物载体(MNA-DPD)自制。马蹄形梯度磁场(0.1T)；恒冷箱切片机(Leica, CM-1800, USA)等。

三、研究方法

实验动物随机分为5组：正常组(n=20)，处理组(n=20)：溶剂对照组(dimethyl sulphoxide group, DMSO)、纳米磁性蛋白组(magnetic nanoparticle-albumin, MNA)、2-2' 联吡啶组(2, 2'-dipyridyl, DPD)和纳米磁性药物载体组(magnetic nanoparticle-albumin-2, 2'-dipyridyl, MNA-DPD)。四个处理组DCVS模型采用二次注血法制作[3]，造模后每天分别通过腰大池给予0.01% DMSO(溶剂)、MNA、DPD和MNA-DPD，剂量50 mg/kg。MNA-DPD和MNA组给予外加磁场磁导向(0.1T)。给药第7天分批处死动物，取基底动脉行HE染色并测定管径，同时行普鲁士蓝染色。Western Blot蛋白定量观察第7天PCNA、VEGF、c-Myc和p53蛋白表达水平。

四、统计学处理

图1 各组基底动脉直径量化和基底动脉壁厚度量化变化 (HE, ×40)
Fig 1 Quantification of basilar artery diameter and basilar artery wall thickness were performed in each group (HE, ×40)
A: Normal basilar artery morphology in sham group; B: Basilar artery spasm in DMSO group; C: Basilar artery spasm in MNA group; D: Basilar artery spasm was slightly improved in DPD group; E: Basilar artery spasm was improved significantly in MNA-DPD group; F: Quantification histogram of basilar artery diameter in each group; G: Histogram of basilar artery wall thickness.

aPlt;0.05,vsMNA group;bPlt;0.05,vsDPD group.

Bar=100 μm.

结 果

一、HE染色

第7天时：对照组正常基底动脉形态，DMSO组基底动脉明显痉挛，MNA组基底动脉明显痉挛，DPD组基底动脉痉挛稍有改善，MNA-DPD组基底动脉痉挛明显改善，呈现差异显著(Plt;0.05)。各组基底动脉直径量化和基底动脉壁厚度量化变化见图1(HE, ×40)。

二、普鲁士蓝染色

40倍镜下DMSO组、MNA组基底动脉明显痉挛，MNA-DPD组基底动脉基本正常的形态。并可见到达蛛网膜下腔的纳米磁性微粒被染成蓝色(铁离子)(见图2)。Western Blot第7天时模型基底动脉PCNA、P53、c-Myc和VEGF蛋白在DMSO组、MNA组和DPD组呈现高表达，MNA-DPD组呈现低表达，MNA-DPD组与DPD组比较有显著差异(Plt;0.05)，MNA-DPD与MNA组比较有显著差异(Plt;0.05，图3)。

图2 各组血管普鲁士蓝染色结果(×40)
Fig 2 Prussian blue staining results for each group of blood vessels (×40)
A: The basilar artery in sham group showed the morphology; B: Basilar artery spasm in DMSO group; C: Basilar artery spasm in MNA group; D: Basilar artery spasm was slightly improved in DPD group; E: Basilar artery spasm was improved significantly in MNA-DPD group.

Bar=100 μm.

图3 基底动脉中PCNA、P53、c-Myc和VEGF的Western Blot定量分析
Fig 3 Western Blot analysis of PCNA, P53, c-Myc and VEGF in basilar artery
A: Western Blot analysis of PCNA in basilar artery; B: Western Blot analysis of P53 in basilar artery; C: Gray scale ratio of PCNA compared with actin control; D: Gray scale ratio of P53 compared with actin control; E: Western Blot analysis of c-Myc in basilar artery; F: Western Blot analysis of VEGF in basilar artery; G: Gray scale ratio of c-Myc compared with actin control; H: Gray scale ratio of VEGF compared with actin control.

aPlt;0.05,vsMNA group;bPlt;0.05,vsDPD group.

讨 论

迟发性脑血管痉挛(DCVS)机制错综复杂，治疗方法众多，但疗效甚微[4-6]。目前流行的DCVS治疗是应用Ca2+拮抗剂(如尼莫通等)，价格昂贵且临床疗效不理想，因此DCVS治疗的途径或体系值得分析考虑。本研究根据DCVS相对肯定的病理机制，经过DCVS模型腰大池途径，利用磁导向将纳米磁性药物载体(MNA-DPD)靶向聚集于DCVS部位，并缓释药物，大大增加药物的有效浓度。同时还具有抗氧化、抗自由基(阻止Fe2+参与自由基的形成)、抗免疫、抗增殖和抗细胞水肿的作用。抑制内皮细胞和平滑肌细胞的异常增殖和/或迁移，防止脑血管的痉挛后期出现的器质性痉挛[7-9]。

实验证明MNA-DPD中，MNA具有良好的生物相溶性、水溶性、磁响应性、稳定性和靶向性[10-11]。DPD对Fe2+有高度亲和力，可以螯合Fe2+。MNA-DPD可以在细胞内外通过药物浓度梯度或者在外在磁场作用下缓慢释放DPD。通过腰大池给药给予MNA-DPD，在外在聚焦梯度磁场作用下，由于纳米磁性药物载体具有磁响应性和靶向性，可以通过脑脊液循环靶向聚集于位局部出血的蛛网膜下腔，缓慢释放DPD，DPD 通过其螯合环结合OxyHb降解的 Fe2+，阻断Fe2+导致DCVS的一系列病理过程，有效阻止脑血管痉挛的发生；铁离子螯合剂还可以减轻脑水肿，减轻OxyHb对平滑肌的副作用[12]。

c-Myc和P53基因表达与PCNA有明显正相关性，是促进血管平滑肌细胞表型转换及增殖，加速新生内膜形成的重要原因。VEGF在新生内膜形成中所起的作用最为重要，VEGF还是血管平滑肌细胞重要的化学趋化因子，促进血管平滑肌细胞增殖。P53还可以促进细胞的凋亡，导致或加重血管痉挛[13]。因此上述研究基本证实了MNA-DPD定向干预DCVS治疗的有效性。

本研究根据DCVS相对肯定的病理机制，经过DCVS模型腰大池途径，利用磁导向将纳米磁性药物载体靶向聚集于DCVS部位，并缓释药物，大大增加药物的有效浓度。铁螯合剂DPD对Fe2+具有的高度和亲和力，能够有效地螯合并清除蛛网膜下腔里OxyHb解离出的Fe2+，阻断DCVS的病理过程，同时还具有抗自由基(Fe2+参与自由基的形成)的作用，最终达到有效地扩张痉挛的脑血管。

1DABABNEH H, GUERRERO W, MEHTA S, et al. Possible role of Eptifibatide drip in-patient with aneurysmal subarachnoid hemorrhage in vasospasm prevention [J]. J Vasc Interv Neurol, 2014, 7(3): 8-13.

2NISHIZAWA S. The roles of early brain injury in cerebral vasospasm following subarachnoid hemorrhage: from clinical and scientific aspects [M]//Cerebral vasospasm: neurovascular events after subarachnoid hemorrhage. Vienna: Springer, 2013: 207-211.

3SPALLONE A, PASTORE F S. Cerebral vasospasm in a double-injection model in rabbit [J]. Surg Neurol, 1989, 32(6): 408-417.

4VATTER H, KONCZALLA J, WEIDAUER S, et al. Effect of delayed cerebral vasospasm on cerebrovascular endothelin A receptor expression and function [J]. J Neurosurg, 2007, 107(1): 121-127.

5HAKAN T, BERKMAN M Z, ERSOY T, et al. Anti-inflammatory effect of meloxicam on experimental vasospasm in the rat femoral artery [J]. J Clin Neurosci, 2008, 15(1): 55-59.

6O' CONNOR D M, O' BRIEN T. Nitric oxide synthase gene therapy: progress and prospects [J]. Expert Opin Biol Ther, 2009, 9(7): 867-878.

7WU H, WU T, LI M, et al. Efficacy of the lipid-soluble iron chelator 2, 2'-dipyridyl against hemorrhagic brain injury [J]. Neurobiol Dis, 2012, 45(1): 388-394.

8YU Y, LIN Z, YIN Y, et al. The ferric iron chelator 2, 2'-dipyridyl attenuates basilar artery vasospasm and improves neurological function after subarachnoid hemorrhage in rabbits [J]. Neurol Sci, 2014, 35(9): 1413-1419.

9吴一娜, 方亦斌, 韩昭龙, 等. 氢气盐水治疗迟发性脑血管痉挛实验研究 [J]. 中华神经外科疾病研究杂志, 2014, 13(6): 491-494.

10CHENG Y, MORSHED R A, AUFFINGER B, et al. Multifunctional nanoparticles for brain tumor imaging and therapy [J]. Adv Drug Deliv Rev, 2014(66): 42-57.

11CHERTOK B, DAVID A E, YANG V C. Polyethyleneimine-modified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration [J]. Biomaterials, 2010, 31(24): 6317-6324.

12MÉTHY D, BERTRAND N, PRIGENT-TESSIER A, et al. Beneficial effect of dipyridyl, a liposoluble iron chelator against focal cerebral ischemia: in vivo and in vitro evidence of protection of cerebral endothelial cells [J]. Brain Res, 2008 (1193): 136-142.

13YUKSEL S, TOSUN Y B, CAHILL J, et al. Early brain injury following neurysmal subarachnoid hemorrhage: emphasis on cellular apoptosis. Early brain injury following aneurysmal subarachnoid hemorrhage: emphasis on cellular apoptosis [J]. Turk Neurosurg, 2012, 22(5): 529-533.

Theinterventionaleffectofmagneticnanoparticle-albumin-2, 2'-dipyridyl(MNA-DPD)ondelayedcerebralvasospasm

WANGZiwen1,GUIZheng1,LUXuegang2,GUODong1,ZHUMing1,LIZhen1,HANXinwei1,YUYaoyu1

1DepartmentofInterventionalRadiology,FirstAffiliatedHospitalofZhengzhouUniversity,Zhengzhou450001;2DepartmentofMaterialPhysics,SchoolofScience,Xi'anJiaotongUniversity,Xi'an710049, China

ObjectiveThe interventional effect of magnetic nanoparticle-albumin-2, 2'-dipyridyl (MNA-DPD) on delayed cerebral vasospasm (DCVS) with magnetic field targeting was explored.MethodsThe experimental animals, 100 healthy adult New Zealand rabbits, were randomly divided into 5 groups: dimethyl sulphoxide group (DMSO), magnetic nanoparticle- albumin group (MNA), 2, 2'-dipyridyl group (DPD), magnetic nanoparticle-albumin-2, 2'-dipyridyl group (MNA-DPD), and control group. DCVS models were made by the secondary injection of blood in DMSO group, MNA group, DPD group and MNA-DPD group. 0.01% DMSO, MNA, DPD and MNA-DPD were administered respectively in each experimental group once every day (50 mg/kg). Focusing magnetic field was guided in MNA group and MNA-DPD group. In 7 d after injection, animals were killed and given perfusion fixation. Basilar arteries were extracted for HE staining, Prussian blue staining and Western Blot test. The expression of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), c-Myc protein and Tumor protein p53 in the seventh day were measured with Western Blot test.ResultsAt 7 d after injection, HE staining and Prussian blue staining showed obvious vasospasm in DMSO group and MNA group, moderate vasospasm in DPD group and nearly no vasospasm in MNA-DPD group. It was significantly different in MNA-DPD group compared with MNA group, DPD group and DMSO group (Plt;0.05). Prussian blue staining showed magnetic nanoparticles were dyed blue in the subarachnoid space of MNA group and MNA-DPD group. Western Blot test indicated high expression of PCNA, P53, c-Myc and VEGF in DMSO group, MNA group and DPD group, while low expression in MNA-DPD group (Plt;0.05).ConclusionMNA-DPD injection via lumbar cistern can effectively relieve DCVS in rabbits with magnetic field targeting.

MNA-DPD; Delayed cerebral vasospasm; Subarachnoid hemorrhage

1671-2897(2017)16-293-04

R 743.35

A

国家自然科学基金资助项目(81171116)

汪子文，硕士，住院医师，E-mail: 492047008@qq.com

*通讯作者：于耀宇，副教授、主任医师，E-mail: yuyaoyu666@aliyun.com

2016-08-30；

2016-12-25)