[1]杨倩,蔡雯婷,金惠子,等.聚乙烯亚胺修饰的荧光素钠纳米颗粒在荧光素眼底血管造影术的应用和安全性[J].眼科新进展,2020,40(11):1005-1010.[doi:10.13389/j.cnki.rao.2020.0225]
　YANG Qian,CAI Wenting,JIN Huizi,et al.Applicationand safetyof polyethyleneimine modified fluorescein sodium nanoparticles in fundus fluorescein angiography[J].Recent Advances in Ophthalmology,2020,40(11):1005-1010.[doi:10.13389/j.cnki.rao.2020.0225]
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聚乙烯亚胺修饰的荧光素钠纳米颗粒在荧光素眼底血管造影术的应用和安全性/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年11期
页码:
1005-1010
栏目:
实验研究
出版日期:
2020-11-05

文章信息/Info

Title:
Applicationand safetyof polyethyleneimine modified fluorescein sodium nanoparticles in fundus fluorescein angiography
作者:
杨倩; 蔡雯婷; 金惠子; 余咚卉; 沈天怡; 于靖
230032　安徽省合肥市，安徽医科大学上海临床学院（杨倩，于靖）；200072　上海市，同济大学附属第十人民医院眼科（蔡雯婷，金惠子，余咚卉，沈天怡）
Author(s):
YANG Qian1; CAI Wenting2; JIN Huizi2; YU Donghui2; SHEN Tianyi2; YU Jing1
1.Shanghai Clinical College，Anhui Medical University,Hefei 230032,Anhui Province，China
2.Department of Ophthalmology,Shanghai Tenth People’s Hospital Affiliated to Tongji University,Shanghai 200072,China
关键词:
荧光素钠; 荧光素眼底血管造影术; 聚乙烯亚胺; 造影剂
Keywords:
fluorescein sodium;  fundus fluorescein angiography;  polyethyleneimine;  contrast agents
分类号:
R770.4
DOI:
10.13389/j.cnki.rao.2020.0225
文献标志码:
A
摘要:
目的　探讨聚乙烯亚胺修饰的荧光素钠（PEI-NHAc-FS）纳米颗粒（nanoparticles,NPs）在荧光素眼底血管造影术的应用和安全性。方法　选取90只健康棕色雄性挪威大鼠作为实验动物。将1 mL 荧光素钠（200 g·L-1）加入10 mol的1-乙基-（3-二甲基氨基丙基）碳酰二亚胺盐酸盐（EDC）混合物中搅拌30 min，再加入10 mol的N-羟基琥珀酰亚胺搅拌3 h。然后将该溶液加入到5 mL聚乙烯亚胺（polyethyleneimine,PEI）（76 g·L-1）中，剧烈搅拌3 d获得PEI-NH2-FS。将2 mL三乙胺加入到PEI-NH2-FS原液中，30 min后再将1 mL的乙酸酐加入混合物中搅拌24 h，合成PEI-NHAc-FS。使用核磁共振波谱仪和紫外可见光吸收光谱观察PEI-NHAc-FS的结构，并对合成的NPs结构进行表征；CCK-8检测其细胞毒性。选取30只大鼠，根据荧光素钠和PEI-NHAc-FS NPs的浓度（5 g·L-1、10 g·L-1、50 g·L-1）将大鼠分为6组，每组5只，分别于注射前及注射后5 min、20 min、30 min和60 min进行眼底摄像，采集大鼠眼底血管荧光素图像。用激光诱导大鼠脉络膜新生血管模型后，取10只大鼠，随机分为10 g·L-1荧光素钠组和PEI-NHAc-FS NPs组，每组5只，分别于注射前及注射后5 min、20 min、30 min和60 min通过眼底血管造影成像观察病灶处渗漏。另取40只大鼠，随机分为10 g·L-1荧光素钠组和PEI-NHAc-FS NPs组，每组20只，于注射后10 min、20 min、30 min和60 min进行荧光冰冻切片。将10只大鼠随机分为对照组（注射生理盐水）和PEI-NHAc-FS NPs组，每组5只，行组织切片HE染色和视网膜电图检测。采用HE染色和视网膜电图观察PEI-NHAc-FS NPs在体内的生物安全性。结果　通过核磁共振和紫外可见光吸收光谱观察到荧光素钠与PEI成功耦合。发射荧光光谱显示，游离荧光素钠和PEI-NHAc-FS NPs的最大发射波长分别出现在546 nm和544 nm处。CCK-8检测结果表明，不同浓度的荧光素钠和PEI-NHAc-FS NPs处理人脐静脉内皮细胞12 h、24 h后，细胞活性差异无统计学意义（P>0.05）。即使用10 μmol·L-1 PEI-NHAc-FS NPs处理24 h和未处理之间细胞存活率差异亦无统计学意义（P>0.05）。在5 g·L-1游离荧光素钠组和PEI-NHAc-FS NPs组中，均仅显影视网膜主要血管，不能用于荧光素眼底血管造影的诊断。而在10 g·L-1游离荧光素钠组和PEI-NHAc-FS NPs组可清晰地显示视网膜主要血管及微血管的结构，且在注射后30 min时血管荧光强度基本相同。PEI-NHAc-FS NPs组在60 min时荧光强度已基本消失，而游离荧光素钠组仍保持较强的荧光强度。10 g·L-1游离荧光素钠组和PEI-NHAc-FS NPs组荧光素眼底血管造影结果显示，经尾静脉注射造影剂后，视网膜血管内可见荧光成像；同时病灶部位可见荧光渗漏。游离荧光素钠组在视网膜组织中荧光较强，对视网膜组织有较强的吸附和渗透作用，而PEI-NHAc-FS NPs组在视网膜组织中荧光较弱，对视网膜组织吸附较弱。HE染色和视网膜电图对造影剂进行体内生物安全性分析结果显示，PEI-NHAc-FS NPs安全性较好。结论　PEI-NHAc-FS NPs能够安全、有效地用于荧光素眼底血管造影。
Abstract:
Objective　To investigatethe application and safety of fluorescein sodium (PEI-NHAc-FS) nanoparticles (NPs) modified with polyethyleneimine in fundus fluorescein angiography (FFA).Methods　Ninety healthy brown male brown Norway rats were used as experimental animals. We took 1 mL fluorescein sodium (200 g·L-1) in 10 mol of 1-ethyl-3-(3-dimethylaminopropy) carbodiimide hydrochloride mixture and stirred for 30 minutes, followed by the addition of 10 mol equivalents of the N-hydroxysuccinimide and stirring for another 3 hours.Next, this solution was added into the 5 mL PEI (76 g·L-1)under vigorous stirring for 3 days to obtain PEI-NH2-FS.Then the solution was added to 5 mL PEI (76 g·L-1), and PEI-NH2-FS was obtained by vigorous stirring for 3 days.Briefly, 2 mL triethylamine was added to the raw solution of the PEI-NH2-FS. After 30 minutes, 1 mL acetic anhydride to the remaining primary amines in PEI was added to the mixture and stirred for 24 hours and PEI-NHAc-FS was synthesized. Nuclear magnetic resonance (NMR) spectrometerand ultraviolet-visible spectroscopy was recorded to observe the structure of PEI-NHAc-FS and performed to characterize (NPs). The CCK-8 was used to evaluate the cell cytotoxicity. According to the concentrations of fluorescein sodium and PEI-NHAc-FS NPs (5 g·L-1, 10 g·L-1, 50 g·L-1), thirty rats were randomly divided into 6 groups (5 rats in each group) for fundus angiography before and 5 minutes, 20 minutes, 30 minutes and 60 minutes after injection. Laser-induced choroidal neovascularization model was established. Forty rats in 90 rats were randomly divided into 10 g·L-1 fluorescein sodium group and PEI-NHAc-FS NPs group (20 rats in each group), followed by fluorescence frozen section imaging 10 minutes, 20 minutes, 30 minutes and 60 minutes after injection. Ten rats in 90 rats were randomly divided into control group (normal saline was given)and PEI-NHAc-FS NPs group (5 rats in each group). HE staining and electroretinogram were used to observe the biosafety of PEI-NHAc-FS NPs in vivo.Results　The successful coupling of fluorescein sodium with PEI by NMR spectrometer and ultraviolet-visible spectroscopy. The emission fluorescence spectra showed that the maximum emission wavelength of fluorescein sodium and PEI-NHAc-FS NPs appeared at 546 nm and 544 nm, respectively. The results of CCK-8 showed that there was no significant difference in cell activity between HUVECs cells treated with different concentrations of fluorescein sodiumand PEI-NHAc-FS NPs for 12 hours and 24 hours (P>0.05). Even when 10 μmol·L-1 PEI-NHAc-FS was treated with NPs for 24 hours and untreated, there was no statistically significant difference in cell survival(P>0.05). In the 5 g·L-1 free fluorescein sodium group and PEI-NHAc-FS NPs group，only images of the main retinal vessels were recorded in both groups，which could not be used for the diagnosis of FFA.However, in the 10 g·L-1 free fluorescein sodium group and PEI-NHAc-FS NPs group, the structure of main retinal vessels and retinal microvessels could be clearly displayed.Meanwhile, the fluorescence intensity of vessels was approximately the same up to 30 minutes after injection.The fluorescence intensity of PEI-NHAC-FS NPs group basically disappeared at 60 minutes, while the free fluorescein sodium groups treated with the same FS concentration maintained stronger fluorescence intensity.The FFA was conducted in 10 g·L-1 free FS group or 10 g·L-1 PEI-NHAc-FS NPs group, respectively. The results showed that ...(PLEASE CHECK THE PDF FILE)Conclusion　PEI-NHAc-FS NPs can be used safely and effectively in FFA.

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备注/Memo

备注/Memo:
国家自然科学基金资助(编号：81470648)；中央高校基本科研业务费专项资金（编号：22120180509）
更新日期/Last Update: 2020-11-05