磁導向納米磁性藥物載體對遲發(fā)型腦血管痙攣干預的實驗研究

汪子文 桂錚 盧學剛 郭棟 朱明 李臻 韓新巍 于耀宇

(1鄭州大學第一附屬醫(yī)院介入科，河南 鄭州 450001; 2西安交通大學理學院材料物理系，陜西 西安 710049)

·腦血管疾病診治研究·

磁導向納米磁性藥物載體對遲發(fā)型腦血管痙攣干預的實驗研究

汪子文1桂錚1盧學剛2郭棟1朱明1李臻1韓新巍1于耀宇1*

(1鄭州大學第一附屬醫(yī)院介入科，河南 鄭州 450001;2西安交通大學理學院材料物理系，陜西 西安 710049)

目的探討在磁導向下納米磁性藥物載體(MNA-DPD)對遲發(fā)型腦血管痙攣(DCVS)干預的有效性。方法健康成年新西蘭白兔100只，隨機分為5組：正常組(n=20)，處理組(n=20)：溶劑對照組(DMSO)、納米磁性蛋白組(MNA)、2-2'聯(lián)吡啶組(DPD)和納米磁性藥物載體組(MNA-DPD)。處理組采用二次注血法制作DCVS模型，然后每天分別通過腰大池給予0.01% DMSO、MNA、DPD和MNA-DPD，劑量50 mg/kg。MNA組、MNA-DPD組給予外加磁場磁導向。在給藥7 d時處死動物，取其基底動脈行HE染色并測定管徑，同時行普魯士藍染色。Western Blot定量分析給藥后7 d動物模型基底動脈增殖細胞核抗原(PCNA)、血管內(nèi)皮生長因子(VEGF)、c-Myc蛋白和p53蛋白表達水平。結(jié)果HE染色和普魯士藍染色顯示DMSO組、MNA組基底動脈明顯痙攣，DPD組基底動脈可見痙攣，MNA-DPD組基底動脈基本正常的形態(tài)(Plt;0.05)。并可見蛛網(wǎng)膜下腔的納米磁性微粒被染成藍色。Western Blot定量分析PCNA、p53、c-Myc、VEGF蛋白在DMSO組、MNA組和DPD組呈現(xiàn)高表達，MNA-DPD組呈現(xiàn)低表達(Plt;0.05)。結(jié)論在磁導向下，腰大池給予納米磁性藥物載體，能夠有效地抑制DCVS。

磁導向納米磁性藥物載體; 遲發(fā)性腦血管痙攣; 蛛網(wǎng)膜下腔出血

自發(fā)性蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)85%以上是腦動脈瘤破裂所致，遲發(fā)性腦血管痙攣(delayed cerebral vasospasm, DCVS)是SAH后病情加重，致殘、致死的主要原因。約70%以上SAH出現(xiàn)DCVS[1-2]。目前流行的DCVS治療是應用Ca2+拮抗劑，療效不理想，至今仍存在爭議。本研究根據(jù)DCVS相對肯定的病理機制，經(jīng)過DCVS模型腰大池途徑，利用磁導向?qū)⒓{米磁性藥物載體靶向聚集于DCVS部位緩釋藥物，增加藥物的有效濃度。有效改善并阻止DCVS病理進程。

材料與方法

一、實驗動物

健康成年新西蘭大白兔100只 (鄭州大學實驗動物中心提供), 雌雄不限，平均體重2.5 kg。

二、實驗試劑和儀器

兔抗抑癌基因p53抗體、兔抗血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)抗體、小鼠抗增殖細胞核抗原(proliferating cell nuclear antigen, PCNA)抗體和兔抗原癌基c-myc抗體(Chemicon, Temecula, CA, USA)；蘇木素伊紅染色試劑盒(中國碧云天)；納米磁性藥物載體(MNA-DPD)自制。馬蹄形梯度磁場(0.1T)；恒冷箱切片機(Leica, CM-1800, USA)等。

三、研究方法

實驗動物隨機分為5組：正常組(n=20)，處理組(n=20)：溶劑對照組(dimethyl sulphoxide group, DMSO)、納米磁性蛋白組(magnetic nanoparticle-albumin, MNA)、2-2' 聯(lián)吡啶組(2, 2'-dipyridyl, DPD)和納米磁性藥物載體組(magnetic nanoparticle-albumin-2, 2'-dipyridyl, MNA-DPD)。四個處理組DCVS模型采用二次注血法制作[3]，造模后每天分別通過腰大池給予0.01% DMSO(溶劑)、MNA、DPD和MNA-DPD，劑量50 mg/kg。MNA-DPD和MNA組給予外加磁場磁導向(0.1T)。給藥第7天分批處死動物，取基底動脈行HE染色并測定管徑，同時行普魯士藍染色。Western Blot蛋白定量觀察第7天PCNA、VEGF、c-Myc和p53蛋白表達水平。

四、統(tǒng)計學處理

圖1 各組基底動脈直徑量化和基底動脈壁厚度量化變化 (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.

結(jié) 果

一、HE染色

第7天時：對照組正?；讋用}形態(tài)，DMSO組基底動脈明顯痙攣，MNA組基底動脈明顯痙攣，DPD組基底動脈痙攣稍有改善，MNA-DPD組基底動脈痙攣明顯改善，呈現(xiàn)差異顯著(Plt;0.05)。各組基底動脈直徑量化和基底動脈壁厚度量化變化見圖1(HE, ×40)。

二、普魯士藍染色

40倍鏡下DMSO組、MNA組基底動脈明顯痙攣，MNA-DPD組基底動脈基本正常的形態(tài)。并可見到達蛛網(wǎng)膜下腔的納米磁性微粒被染成藍色(鐵離子)(見圖2)。Western Blot第7天時模型基底動脈PCNA、P53、c-Myc和VEGF蛋白在DMSO組、MNA組和DPD組呈現(xiàn)高表達，MNA-DPD組呈現(xiàn)低表達，MNA-DPD組與DPD組比較有顯著差異(Plt;0.05)，MNA-DPD與MNA組比較有顯著差異(Plt;0.05，圖3)。

圖2 各組血管普魯士藍染色結(jié)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.

圖3 基底動脈中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.

討 論

遲發(fā)性腦血管痙攣(DCVS)機制錯綜復雜，治療方法眾多，但療效甚微[4-6]。目前流行的DCVS治療是應用Ca2+拮抗劑(如尼莫通等)，價格昂貴且臨床療效不理想，因此DCVS治療的途徑或體系值得分析考慮。本研究根據(jù)DCVS相對肯定的病理機制，經(jīng)過DCVS模型腰大池途徑，利用磁導向?qū)⒓{米磁性藥物載體(MNA-DPD)靶向聚集于DCVS部位，并緩釋藥物，大大增加藥物的有效濃度。同時還具有抗氧化、抗自由基(阻止Fe2+參與自由基的形成)、抗免疫、抗增殖和抗細胞水腫的作用。抑制內(nèi)皮細胞和平滑肌細胞的異常增殖和/或遷移，防止腦血管的痙攣后期出現(xiàn)的器質(zhì)性痙攣[7-9]。

實驗證明MNA-DPD中，MNA具有良好的生物相溶性、水溶性、磁響應性、穩(wěn)定性和靶向性[10-11]。DPD對Fe2+有高度親和力，可以螯合Fe2+。MNA-DPD可以在細胞內(nèi)外通過藥物濃度梯度或者在外在磁場作用下緩慢釋放DPD。通過腰大池給藥給予MNA-DPD，在外在聚焦梯度磁場作用下，由于納米磁性藥物載體具有磁響應性和靶向性，可以通過腦脊液循環(huán)靶向聚集于位局部出血的蛛網(wǎng)膜下腔，緩慢釋放DPD，DPD 通過其螯合環(huán)結(jié)合OxyHb降解的 Fe2+，阻斷Fe2+導致DCVS的一系列病理過程，有效阻止腦血管痙攣的發(fā)生；鐵離子螯合劑還可以減輕腦水腫，減輕OxyHb對平滑肌的副作用[12]。

c-Myc和P53基因表達與PCNA有明顯正相關性，是促進血管平滑肌細胞表型轉(zhuǎn)換及增殖，加速新生內(nèi)膜形成的重要原因。VEGF在新生內(nèi)膜形成中所起的作用最為重要，VEGF還是血管平滑肌細胞重要的化學趨化因子，促進血管平滑肌細胞增殖。P53還可以促進細胞的凋亡，導致或加重血管痙攣[13]。因此上述研究基本證實了MNA-DPD定向干預DCVS治療的有效性。

本研究根據(jù)DCVS相對肯定的病理機制，經(jīng)過DCVS模型腰大池途徑，利用磁導向?qū)⒓{米磁性藥物載體靶向聚集于DCVS部位，并緩釋藥物，大大增加藥物的有效濃度。鐵螯合劑DPD對Fe2+具有的高度和親和力，能夠有效地螯合并清除蛛網(wǎng)膜下腔里OxyHb解離出的Fe2+，阻斷DCVS的病理過程，同時還具有抗自由基(Fe2+參與自由基的形成)的作用，最終達到有效地擴張痙攣的腦血管。

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吳一娜, 方亦斌, 韓昭龍, 等. 氫氣鹽水治療遲發(fā)性腦血管痙攣實驗研究 [J]. 中華神經(jīng)外科疾病研究雜志, 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

國家自然科學基金資助項目(81171116)

汪子文，碩士，住院醫(yī)師，E-mail: 492047008@qq.com

*通訊作者：于耀宇，副教授、主任醫(yī)師，E-mail: yuyaoyu666@aliyun.com

2016-08-30；

2016-12-25)