Article(id=1198652629552300593, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0011, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1672848000000, receivedDateStr=2023-01-05, revisedDate=1683907200000, revisedDateStr=2023-05-13, acceptedDate=null, acceptedDateStr=null, onlineDate=1763710656774, onlineDateStr=2025-11-21, pubDate=1691769600000, pubDateStr=2023-08-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763710656774, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763710656774, creator=13701087609, updateTime=1763710656774, updator=13701087609, issue=Issue{id=1198652605778985059, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='8', pageStart='0', pageEnd='2540', issueExtLink='null', onlineDate='null', pubDate='1691769600000', pubDateStr='2023-08-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763710651106, creator='13701087609', updateTime=1763710739504, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1198652976601596347, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198652976601596348, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198652605778985059, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2512, endPage=2521, ext={EN=ArticleExt(id=1198652630353412719, articleId=1198652629552300593, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Preparation of mesoporous silica nanoparticles with different sizes and study on the correlation between size and toxicity, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

To investigate the crucial role of particle size in the biological effects of nanoparticles, a series of mesoporous silica nanoparticles (MSNs) were prepared with particle size gradients (50, 100, 150, 200 nm) with the traditional Stober method and adjusting the type and ratio of the silica source. The correlation between toxicity and size-caused biological effects were then further examined both in vitro and in vivo. The results indicated that the prepared MSNs had a uniform size, good dispersal, and ordered mesoporous structure. Hemolytic toxicity was found to be independent of particle size. At the cellular level, MSNs with smaller particle sizes were more readily internalized by cells, which initiated to more intense oxidative stress, therefor inducing higher cytotoxicity, and apoptosis rate. In vivo studies demonstrated that MSNs primarily accumulated in the liver and kidneys of mice. Pharmacokinetic analysis revealed that larger MSNs were eliminated more efficiently by the urinary system than smaller MSNs. The mice's body weight monitoring, blood tests, and pathological sections of major organs indicated good biocompatibility for MSNs of different sizes. Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Zhejiang Chinese Medical University. Overall, this study prepared MSNs with a particle size gradient to investigate the correlation between toxicity and particle size using macrophages and endothelial cells. The study also examined the biosafety of MSNs with different particle sizes in vivo and in vitro, which could help to improve the safety design strategy of MSNs for drug delivery systems.

, authors=null, authorsList=Xiao-wei XIE, Meng-ying CHENG, Wei-xiang FANG, Xue LIN, Wen-ting GU, Kai-ling YU, Ting-xian YE, Wei-yi CHENG, Li HE, Hang-sheng ZHENG, Ying-hui WEI, Ji-gang PIAO, Fan-zhu LI, authorCompany=null, correspAuthors=Ji-gang PIAO, Fan-zhu LI, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2023 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1198652632928715544, articleId=1198652629552300593, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=不同粒径介孔二氧化硅纳米颗粒的制备及其与毒性的相关性研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

为探究粒径对纳米粒生物学效应的关键作用, 本研究采用传统Stober法以及通过调整硅源种类以及比例制备系列粒度梯度(50、100、150、200 nm) 介孔二氧化硅纳米粒(mesoporous silica nanoparticles, MSNs), 并在体内外探究了其毒性与粒径产生的生物学效应的关联性。结果表明, 所制备的各组MSNs粒径大小均一、水分散性良好, 且具备有序的介孔结构; 溶血毒性与粒径无相关性; 细胞水平上, 小粒径的MSNs更有利于细胞内化, 引发更加强烈的氧化应激反应, 诱导更高的细胞毒性和细胞凋亡率; 体内研究结果表明MSNs主要蓄积在小鼠肝脏与肾脏; 药动学结果显示, 大粒径MSNs较小粒径MSNs更易经泌尿系统消除; 小鼠体重监测、血常规及主要脏器病理切片结果说明不同粒径MSNs均具有良好的生物相容性。所有动物实验经浙江中医药大学实验动物伦理委员会审查[批准号: SYXK (浙) 2021-0012], 符合实验动物伦理相关规范。综上所述, 本研究制备粒度梯度的MSNs对比探讨巨噬细胞和内皮细胞的毒性-粒径相关关系, 并进一步从体内外分别考察不同粒径MSNs的生物安全性, 有助于完善MSNs应用于递药系统的安全性设计策略。

, authors=null, authorsList=谢晓伟, 程梦莹, 方威祥, 林雪, 顾文婷, 余凯玲, 叶婷娴, 程苇钇, 何莉, 郑杭生, 魏颖慧, 朴寄纲, 李范珠, authorCompany=null, correspAuthors=朴寄纲, 李范珠, authorNote=null, correspAuthorsNote=
*朴寄纲, E-mail: ;
李范珠, Tel: 86-571-61768130, E-mail:
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The activity of lactic dehydrogenase (C) in RAW264.7 and HUVEC treated with different size; superoxide dismutase activity (D) in RAW264.7 and HUVEC treated with different size MSNs. <i>n</i> = 3, <sup>∆</sup><i>P</i> < 0.05, <sup>∆∆</sup><i>P</i> < 0.01 <i>vs</i> 50 nm MSNs group; <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> 100 nm MSNs group; <sup>**</sup><i>P</i> < 0.01 <i>vs</i> 150 nm MSNs group. 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不同粒径介孔二氧化硅纳米颗粒的制备及其与毒性的相关性研究
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谢晓伟 , 程梦莹 , 方威祥 , 林雪 , 顾文婷 , 余凯玲 , 叶婷娴 , 程苇钇 , 何莉 , 郑杭生 , 魏颖慧 , 朴寄纲 * , 李范珠 *
药学学报 | 研究论文 2023,58(8): 2512-2521
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药学学报 |研究论文 2023 , 58 (8) : 2512 -2521
不同粒径介孔二氧化硅纳米颗粒的制备及其与毒性的相关性研究
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谢晓伟, 程梦莹, 方威祥, 林雪, 顾文婷, 余凯玲, 叶婷娴, 程苇钇, 何莉, 郑杭生, 魏颖慧, 朴寄纲* , 李范珠*
作者信息
  • 浙江中医药大学药学院, 浙江 杭州 310000
通讯作者:
*朴寄纲, E-mail: ;
李范珠, Tel: 86-571-61768130, E-mail:
Preparation of mesoporous silica nanoparticles with different sizes and study on the correlation between size and toxicity
Xiao-wei XIE, Meng-ying CHENG, Wei-xiang FANG, Xue LIN, Wen-ting GU, Kai-ling YU, Ting-xian YE, Wei-yi CHENG, Li HE, Hang-sheng ZHENG, Ying-hui WEI, Ji-gang PIAO* , Fan-zhu LI*
Affiliations
  • College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310000, China
出版时间: 2023-08-12 doi: 10.16438/j.0513-4870.2023-0011
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为探究粒径对纳米粒生物学效应的关键作用, 本研究采用传统Stober法以及通过调整硅源种类以及比例制备系列粒度梯度(50、100、150、200 nm) 介孔二氧化硅纳米粒(mesoporous silica nanoparticles, MSNs), 并在体内外探究了其毒性与粒径产生的生物学效应的关联性。结果表明, 所制备的各组MSNs粒径大小均一、水分散性良好, 且具备有序的介孔结构; 溶血毒性与粒径无相关性; 细胞水平上, 小粒径的MSNs更有利于细胞内化, 引发更加强烈的氧化应激反应, 诱导更高的细胞毒性和细胞凋亡率; 体内研究结果表明MSNs主要蓄积在小鼠肝脏与肾脏; 药动学结果显示, 大粒径MSNs较小粒径MSNs更易经泌尿系统消除; 小鼠体重监测、血常规及主要脏器病理切片结果说明不同粒径MSNs均具有良好的生物相容性。所有动物实验经浙江中医药大学实验动物伦理委员会审查[批准号: SYXK (浙) 2021-0012], 符合实验动物伦理相关规范。综上所述, 本研究制备粒度梯度的MSNs对比探讨巨噬细胞和内皮细胞的毒性-粒径相关关系, 并进一步从体内外分别考察不同粒径MSNs的生物安全性, 有助于完善MSNs应用于递药系统的安全性设计策略。

介孔二氧化硅纳米粒  /  粒径  /  毒性-粒径相关性  /  氧化应激  /  纳米毒理

To investigate the crucial role of particle size in the biological effects of nanoparticles, a series of mesoporous silica nanoparticles (MSNs) were prepared with particle size gradients (50, 100, 150, 200 nm) with the traditional Stober method and adjusting the type and ratio of the silica source. The correlation between toxicity and size-caused biological effects were then further examined both in vitro and in vivo. The results indicated that the prepared MSNs had a uniform size, good dispersal, and ordered mesoporous structure. Hemolytic toxicity was found to be independent of particle size. At the cellular level, MSNs with smaller particle sizes were more readily internalized by cells, which initiated to more intense oxidative stress, therefor inducing higher cytotoxicity, and apoptosis rate. In vivo studies demonstrated that MSNs primarily accumulated in the liver and kidneys of mice. Pharmacokinetic analysis revealed that larger MSNs were eliminated more efficiently by the urinary system than smaller MSNs. The mice's body weight monitoring, blood tests, and pathological sections of major organs indicated good biocompatibility for MSNs of different sizes. Animal welfare and the animal experimental protocols were strictly consistent with related ethics regulations of Zhejiang Chinese Medical University. Overall, this study prepared MSNs with a particle size gradient to investigate the correlation between toxicity and particle size using macrophages and endothelial cells. The study also examined the biosafety of MSNs with different particle sizes in vivo and in vitro, which could help to improve the safety design strategy of MSNs for drug delivery systems.

mesoporous silica nanoparticle  /  particle size  /  size-dependent toxicity  /  oxidative stress  /  nanotoxicity
谢晓伟, 程梦莹, 方威祥, 林雪, 顾文婷, 余凯玲, 叶婷娴, 程苇钇, 何莉, 郑杭生, 魏颖慧, 朴寄纲, 李范珠. 不同粒径介孔二氧化硅纳米颗粒的制备及其与毒性的相关性研究. 药学学报, 2023 , 58 (8) : 2512 -2521 . DOI: 10.16438/j.0513-4870.2023-0011
Xiao-wei XIE, Meng-ying CHENG, Wei-xiang FANG, Xue LIN, Wen-ting GU, Kai-ling YU, Ting-xian YE, Wei-yi CHENG, Li HE, Hang-sheng ZHENG, Ying-hui WEI, Ji-gang PIAO, Fan-zhu LI. Preparation of mesoporous silica nanoparticles with different sizes and study on the correlation between size and toxicity[J]. Acta Pharmaceutica Sinica, 2023 , 58 (8) : 2512 -2521 . DOI: 10.16438/j.0513-4870.2023-0011
纳米技术在生物医学领域中快速发展。纳米材料凭借粒径小、毒性低、显著的生物相容性和化学稳定性在生命医学领域展现出卓越的研究价值和巨大的应用潜能[1]。而纳米粒理化性质的差异可能会导致其毒性大相径庭, 阻碍纳米药物的临床运用。其中, 粒径则属于影响其潜在毒性的关键因素[2]。常规尺寸下无毒或低毒性物质, 当其粒径降低至纳米范畴时, 由于表面活性及化学反应性增强, 导致对机体的作用强度及生物效应发生质的改变, 从而引发毒性或增加毒性[3]
介孔二氧化硅纳米粒(mesoporous silica nanoparticles, MSNs) 因兼具纳米粒和介孔材料双重优势而广受研究者们青睐。MSNs拥有生物相容性优异、合成简便、尺寸可控、水分散性良好等特点, 作为极具代表性的药物载体而被广泛运用于纳米递药系统, 并具有极佳的应用前景[4-7]。有研究表明, 随着MSNs粒径的逐渐减小(14~335 nm), MSNs对EAHY926细胞的毒性反应逐渐增强; 此外, 人类支气管肺癌细胞也被发现存在这种MSNs粒径与毒性的相关联系, 但是更大粒径的MSNs似乎并没有细胞毒性作用。因此, 需要进一步从细胞、血液、器官组织等水平上了解MSNs的潜在毒性机制, 并评估其安全性, 更好地科学指导MSNs应用于递药系统的安全性设计。
纳米材料的生物学特性对于研究其组织/器官毒性和生物医学应用具有至关重要的意义[8]。药物进入体内, 是否影响体内正常生理环境是评判药物安全性的重要内容。纳米药物进入体内都会经血液循环进入心血管系统。其中血管内皮细胞, 介于血管壁组织与血液间的一层单核细胞, 作为纳米材料入血后的首道屏障[9]; 另外巨噬细胞, 作为免疫系统中的“第一反应”细胞, 通过吞噬病原体、异物等发挥防御作用, 属于纳米材料入血后的第二道防线[10-12]。因此, 本研究将人脐静脉内皮细胞和巨噬细胞作为研究对象, 探讨不同粒径MSNs对心血管系统的细胞毒性作用。纳米药物体内过程是评价纳米材料生物毒性的另一重要研究内容, 通常包括血液相容性、体内生物分布、血循环周期和生物消除等[13]
本研究制备了分散性良好、性质稳定、具有相似表面特性的四种梯度粒径的MSNs。从细胞水平上探讨不同粒径的MSNs对两种细胞的毒性-粒径相关关系, 后续从体内分布、体液、组织器官水平进一步考察了MSNs的毒性与粒径相关性。根据MSNs的纳米毒性相关评价结果, 有助于控制有害影响的发展趋势并提供规避风险的科学性指导, 以及完善MSNs作为载体在递药系统的安全性设计策略。
药品与试剂  十六烷基三甲基溴化铵(98%, CTAB) (S08J8I37509)、四乙氧基硅烷(98%, TEOS) (BCBV4115)、异硫氰酸荧光素(FITC) (SLCN5721)、3-氨基丙基三乙氧基硅烷(APTES) (MKCR0742)、苏木精和伊红(H & E) 试剂盒, 美国Sigma Aldrich公司; 4', 6-二脒基-2-苯基吲哚(DAPI) (030122220914)、增强型CCK-8试剂盒、Annexin V-FITC/PI细胞凋亡检测试剂盒, 上海碧云天生物技术有限公司; 硅标准品(C10394346), 上海麦克林生化科技有限公司; RPMI-1640培养液(8122742)、胎牛血清(FBS) (2238253), 美国Gibco公司; 青-链霉素溶液(100×) (22328374), Biosharp公司; 乳酸脱氢酶细胞毒性检测ELISA试剂盒、超氧化物歧化酶ELISA试剂盒, 江苏酶免实业有限公司; 二氢乙啶(dihydroethidium, DHE) 荧光探针, 英国Abcam贸易有限公司。
细胞  小鼠单核巨噬细胞白血病细胞(RAW264.7) 购自中国科学院上海细胞库, 人脐静脉内皮细胞(HUMEC) 培养于含有10% FBS、100 IU·mL-1青霉素和100 μg·mL-1链霉素的1640培养基中, 并置于37 ℃、5% CO2培养箱中避光培养。
实验动物  SPF级Balb/c小鼠(上海斯莱克实验动物公司) 4~6周, 体重20 g左右。健康家兔由浙江中医药大学动物实验研究中心提供。动物实验通过浙江中医药大学实验动物管理和伦理委员会批准, 按照浙江中医药大学动物饲养和使用指南进行, 实验动物使用许可证号: SYXK (浙) 2021-0012。
“Stober”法制备MSNs  精密称量模板剂CTAB置于圆底烧瓶中, 依次向瓶内缓慢加入适量TEAH3和去离子超纯水, 70 ℃恒温水浴搅拌1 h至完全溶解, 吸取适量TEOS加入反应液, 70 ℃恒温水浴搅拌4 h至溶液呈淡蓝色乳光, 室温熟化过夜, 反应液15 000 r·min-1离心15 min, 重复2次, 除去残留的CTAB, 将沉淀充分溶解于适量盐酸乙醇溶液(盐酸∶无水乙醇= 1∶30), 置于50 mL圆底烧瓶中80 ℃恒温回流8 h, 更换新的盐酸乙醇溶液后继续回流16 h, 将反应液12 000 r·min-1离心20 min, 弃上清, 并用去离子超纯水与95%乙醇溶液交替洗涤沉淀, 以除去残留的CTAB和盐酸乙醇溶液, 将沉淀分散在少量去离子超纯水中, -80 ℃冷冻过夜, 真空干燥3天, 即得白色MSNs冻干粉末。
粒径、PDI、zeta电位及形态表征  配制一定浓度50、100、150和200 nm粒径的MSNs溶液, 超声5 min, 取1 mL MSNs混悬液置于洁净的样品池中。采用NANO-ZS90 Malvern粒度分析仪分别测定其粒径、PDI和zeta电位; 吸取适量纳米悬液, 滴于100目碳支持膜铜网正面, 干燥后用透射电子显微镜(TEM) 观察纳米粒的外貌形态; 将适量的MSNs粉末用双面铝带固定在黄铜短管上, 表面镀金后采用扫描电子显微镜(SEM) 观察各粒径MSNs的微观形态。
溶血毒性实验  取适量除去纤维蛋白凝块的健康家兔新鲜耳缘静脉血, 加入适量的磷酸盐平衡生理盐水(PBS) 充分混匀后, 1 500 r·min-1离心15 min, 弃上清, 重复3次至上清液呈无色。沉淀红细胞配成2%的红细胞悬液, 并配制系列不同浓度(0.01、0.05、0.1、0.25、0.5、1、2 mg·mL-1) MSNs混悬液, 各组加入等体积的现配红细胞悬液, 另设(蒸馏水) 阳性对照组和(PBS) 阴性对照组, 各管充分混匀后置于37 ℃恒温水浴锅中孵育1 h, 1 500 r·min-1离心15 min, 使用酶标仪在414 nm波长处测定取上清吸光度, 并计算溶血率。
细胞增殖毒性评价  取对数生长期HUVEC和RAW264.7细胞, 分别以每孔4×103个接种于96孔板, 于37 ℃恒温培养箱中培养24 h, 弃去培养液后加入相同的浓度梯度的各粒径MSNs溶液或PBS对照组。分别在培养24和48 h后取出, 弃去培养液, 加入110 µL CCK-8的培养液(CCK-8∶空白1640培养液= 1∶10), 孵育60 min后, 用酶标仪测定450 nm处的吸光度, 并计算细胞存活率。
细胞摄取  取对数生长期的HUVEC和RAW264.7细胞, 分别以7×104个/孔接种于激光共聚焦皿中, 于37 ℃恒温培养箱培养24 h后弃去培养液, 分别加入含10 μmol·L-1 FITC标记的各粒径MSNs的空白1640培养液。孵育4 h后除去培养液, PBS漂洗3次, 4%多聚甲醛固定15 min, 弃去并用PBS漂洗3次; 然后加入含DAPI (0.2 μg·mL-1) 的培养基孵育20 min, PBS轻轻漂洗3次后置于Zeiss LSM880激光共聚焦显微镜下(confocal laser scanning microscope, CLSM) 观察。相同处理办法至细胞孵育完成, 胰酶消化2~5 min, PBS漂洗3次, 800 r·min-1离心5 min, 用PBS将细胞重悬并收集, 另设未用MSNs处理过的空白细胞作为阴性对照组, 用CytoFLEX流式细胞仪定量分析各组细胞摄取率。
细胞凋亡测定  取对数生长期的HUVEC和RAW264.7细胞以5×104个/孔的密度接种于6孔板, 贴壁生长24 h后, 分别加入空白培养基和一系列浓度的含不同粒度梯度MSNs (250和500 μg·mL-1) 培养基培养24 h, 将上层培养液移至适宜离心管中, 预冷的PBS清洗液体加入离心管中, 每孔加入胰酶消化10 min。接着, 加入完全培养基终止消化, 并将消化好的细胞收集至离心管中, 800 r·min-1离心5min, 弃上清。每孔加入200 μL Annexin V-FITC结合液轻轻重悬细胞, 加入10 μL碘化丙啶染色液混匀, 室温避光孵育10~20 min。用PBS将液体补足至约500 μL, 用CytoFLEX流式细胞仪检测不同粒径MSNs对细胞凋亡的影响。
细胞膜完整性损伤  测定乳酸脱氢酶(LDH) 活性, LDH酶活性的定义是每1 mg的蛋白每分钟催化产生1 nmol·L-1丙酮酸, 该值越大表示细胞膜损伤程度越大。吸取10 μL的MSNs处理后的细胞样品上清液加入至96孔板, 并将LDH试剂A与LDH试剂B (氧化型辅酶I溶液, 11.3 mmol·L-1) 按5∶1的比例配制成混合液, 吸取60 μL混合液滴加至每个样品孔, 37 ℃恒温振摇15 min。接着, 加入50 μL LDH试剂C (2, 4-二硝基苯肼溶液) 置于摇床振摇2 min, 后加入150 μL终止试剂, 振摇数次混合均匀后, 室温放置3 min。采用酶标仪在波长450 nm处测定吸光度, 计算LDH酶活性(U·mg-1 prot)。
超氧化物歧化酶(SOD) 活性检测  吸取10 μL的样品上清液加入96孔板, 并按顺序将10 μL酶稀释液、20 μL WST-8溶液和160 μL SOD检测缓冲液依次加入孔内并设置对照组。摇床振摇3 min, 使溶液混匀, 室温静置30 min, 采用酶标仪在波长450 nm处测定吸光度, 计算SOD酶活性(U·mg-1 prot)。
活性氧(ROS) 检测  取对数生长期的HUVEC和RAW264.7细胞以5.0×104个/孔的密度接种于6孔板, 过夜后弃去培养液, PBS轻轻漂洗3次, 每孔加入1 mL不同浓度的(MSNs, 0、100和250 μg·mL-1) 各粒径MSNs溶液, 37 ℃恒温培养24 h, 吸出含药培养液, PBS轻轻漂洗3次, 每孔加入1 mL 10 µmol·L-1新鲜DHE工作液, 37 ℃恒温避光孵育60 min, 吸出工作液, 预冷PBS轻轻漂洗细胞3次, 置于荧光倒置显微镜下观察。
体内分布检测  12只20 ± 2 g的Balb/c小鼠, 随机将其分为4组(n = 3), 通过尾静脉将不同粒径的FITC-MSNs分别注射至小鼠体内(FITC的浓度为2 mg·kg-1)。给药后1、4、8、12、24、42和72 h时, 用异氟烷对小鼠进行麻醉, 置于IVSI小动物活体荧光成像系统中(激发波长450 nm, 发射波长550 nm), 观察不同给药时间后, 不同粒径的FITC-MSNs在体内的分布情况。给药72 h后处死小鼠, 取出主要的器官(心、肝、脾、肺、肾), 用生理盐水清洗后对其荧光强度进行检测。
血液消除监测  12只22 ± 2 g的Balb/c小鼠随机分为4组(n = 3), 通过尾静脉分别注射不同粒径的MSNs, MSNs的浓度为10 mg·kg-1。给药后的小鼠在相同的实验条件下, 在预定的时间点(10 min、30 min、1 h、2 h、4 h、8 h和12 h) 从眼眶采集100 μL血样, 加入含有去离子水(200 μL) 的试管中, 随后加入1 mL 10%硝酸溶液, 放置通风橱至完全硝解, 过0.22 μm滤膜, ICP-MS检测血液中硅元素含量, 考察不同粒径MSNs在血液中随时间消除情况。
尿液消除监测  12只22 ± 2 g的Balb/c小鼠随机分为4组(n = 3), 通过尾静脉注射不同粒径的MSNs, MSNs的浓度为10 mg·kg-1。给药后的小鼠分别放置于代谢笼中, 在预定的时间点(12、24、48和72 h) 采集尿液样本, 随后硝化处理, 过膜后, 用ICP-MS检测尿液中硅元素含量, 考察不同粒径MSNs静脉注射到体内后经尿液随时间的清除情况。
血常规检测  在完成体重检测实验后, 各组随机取3只小鼠, 摘眼球取小鼠全血, 进行血常规检测, 评价小鼠血液安全性。
统计学方法  使用GraphPad Prism 8.0软件进行分析, 数据用$\bar{x}$ ± s表示。数据两组采用t test进行统计比较, P < 0.05认为有显著性差异, P < 0.01认为有差异极显著。
不同粒径MSNs的微观形态TEM和SEM如图 1AB所示, 各尺寸的MSNs呈规则的圆球状, 其真空下的粒径大小符合水合粒径, 基本无粘连, 分散性良好, 各组MSNs呈现出均一规则蜂巢状结构, 可见明显的辐射排列网状介孔。整体而言, 大粒径的MSNs表面看起来相对光滑圆整, 表面孔道较密, 而小粒径的MSNs表面相对粗糙, 表面孔径较小, 孔道较稀疏。
对各组MSNs的粒径、PDI和zeta电位进行测定, 结果如图 1CD所示。各粒径MSNs的分散性指数较低, 说明粒径的均一性良好, 动态光散射法测得的粒径较透射电镜测得值偏大, 推测可能是由于水溶液中, 纳米粒表面-OH结合水中H+形成一层水化层所致; zeta电位的结果显示, 各组MSNs的表面电位均在-22.5~-32.2 mV之间, 且绝对值大于20, 表示具有良好稳定性。
以各粒径MSNs以及其经氨基修饰和FITC标记后的红外光谱图(图 1E) 分析表面官能团。各组分在1 100 cm-1处均有明显吸收峰, 推测是由于Si-O-Si的不对称拉伸震动引起的强烈吸收带; 在470 cm-1附近的吸收峰, 推测为Si-O-Si的弯曲振动吸收峰; 在800 cm-1左右的吸收峰, 推测是Si-O-Si的对称拉伸震动峰; 所有组分均在3 400 cm-1处存在宽峰, 推测是二氧化硅表面的-OH发生伸缩振动产生的; 在NH2-MSNs和FITC-MSNs的红外图谱上, 能观察到在1 550 cm-1附近有一个吸收峰, 推测是因为-NH的弯曲振动形成, 证明-NH2成功修饰在MSNs的表面。FITC-MSNs图谱上看到在1 400 cm-1左右有一个中等吸收峰, 证明了FITC的成功修饰。
各粒径MSNs的氮气吸-脱附等温曲线和孔径分布图(图 1FG) 所示, 四种粒径的二氧化硅纳米粒的氮气吸-脱附等温线均属于IUPAC分类中的IV型分布, 且在相对高压区(P/P0 = 0.8~1.0) 处均有因氮气的毛细管冷凝作用而产生N2滞后环, 以及窄范围内存在尖锐, 这些都是典型的介孔物质吸附特征, 表明所有纳米粒均有介孔结构, 孔道呈圆柱形。50、100、150、200 nm粒径纳米粒的孔径分别为2.06、2.22、3.71和3.98 nm, 孔容积分别为0.605、0.575、1.09和1.13 cm3·g-1, 各组差异不明显, 但是大粒径相对比小粒径的孔容孔径更大, 推测可能表面活性剂和硅源之间的电荷密度比例对粒径和孔径的尺寸均产生一定影响。
经孵育处理后纳米粒的溶血毒性结果所示(图 1H), 各组的溶血率分别为(0.944 ± 0.033) %、(1.789 ± 0.032) %、(4.099 ± 0.129) %、(0.265 ± 0.005) %。根据文献报道, 溶血率不足5%时即可视为没有溶血性[14], 由此可见四种粒径MSNs虽然均具有较好的生物相容性, 组间细微的溶血差异可能是由于粒径因素介导的表面积、吸附力、红细胞膜干扰差异所引起的。
为评价不同尺寸的MSNs对免疫系统和心血管系统造成的影响, 采用CCK-8法测定其对RAW264.7和HUVEC细胞的毒性(图 2AB)。从结果可看出, 经过24 h孵育, 内皮细胞中各组最高给药浓度(500 μg·mL-1) 的细胞存活率分别是(68.35 ± 6.12) %、(73.92 ± 5.09) %、(75.84 ± 6.19) %、(77.26 ± 5.99) %; 巨噬细胞的细胞毒性实验结果可以观察到, 粒径越小的MSNs其细胞毒性越大; 更长时间的孵育条件下, 细胞毒性趋势也保持相同。综上, MSNs所导致的细胞毒性呈现浓度、时间正相关性和粒径负相关性。
考察不同粒径MSNs对细胞摄取的影响; 采用激光共聚焦显微镜初步观察不同粒径MSNs在RAW264.7和HUVEC这两种细胞中的分布情况(图 2CE), 进行较短时间内(4 h) 的考察, 对比在两种细胞中的分布情况, 并没有看出显著的差异; 但是从粒径的角度进行对比, 相同的孵育时间下, 50 nm MSNs的摄取量均显著高于其余三种大粒径组。随后采用流式细胞术进一步对FITC标记的各粒径MSNs摄取入细胞中的荧光强度进行定量分析(图 2DF), 直观地评价两种细胞对不同粒径MSNs的摄取情况, 巨噬细胞对各粒径MSNs的摄取能力均高于内皮细胞对其的摄取能力, 同共聚焦显微镜结果相似, 摄取能力差异不大。在巨噬细胞中, 50 nm MSNs的平均荧光强度分别是100、150、200 nm MSNs细胞平均荧光强度的4.60、5.69、6.37倍; 在内皮细胞中50 nm的平均荧光强度则高于其余三组的2.43倍, 此结果说明小粒径MSNs明显更容易被摄取。
采用流式细胞仪对两种细胞经4 h孵育不同浓度的MSNs进行凋亡检测(图 3AB), RAW264.7结果中, 浓度为250 µg·mL-1时, 50 nm MSNs诱导的总凋亡率为18.04%, 其中晚凋为6.57%, 与其余三组均有显著性差异(P < 0.05); HUVEC也表现出同样的趋势, 50 nm MSNs诱导的凋亡率均明显高于其余三组。从两组细胞凋亡结果推测不同粒径MSNs对细胞凋亡的影响程度会随孵育MSNs的浓度增加而增加, 趋势明显且呈浓度依赖特性; 对比细胞的早凋与晚凋, MSNs诱导的晚凋随粒径的增加呈显著性增加, 推测MSNs主要通过诱导细胞晚期凋亡造成细胞损伤且具有浓度依赖特性。
细胞膜完整性损伤测定结果(图 3C), 50 nm MSNs的LDH释放量均显著高于其余三组, 说明50 nm MSNs对细胞膜的破坏能力显著高于其余三个粒径的MSNs。当浓度达到250 µg·mL-1时, 50 nm组RAW264.7细胞的LDH活性为(40.28 ± 1.77) U·mg-1 prot, 分别是100、150、200 nm组的1.36、1.8、1.67倍; 在HUVEC细胞中, 50 nm组LDH活性为(32.79 ± 0.85) U·mg-1 prot, 分别是其余三组的1.21、1.38、1.39倍, 说明MSNs对细胞类型之间的影响存在差异; 随着MSNs浓度的增加, 各组的LDH释放量均有所增加, 浓度为50 µg·mL-1时, 各组相较对照组LDH活性已有显著性差异。结果显示, 介孔二氧化硅诱导的巨噬细胞和内皮细胞毒性具有剂量相关性且存在细胞类型间差异情况。
SOD活性酶检测结果(图 3D) 与空白组相比, 当浓度为25 µg·mL-1时, 50 nm组显著降低RAW264.7和HUVEC细胞的SOD含量(P < 0.01); 当浓度增至100和250 µg·mL-1时, 其余三组产生的SOD含量在RAW264.7和HUVEC细胞中均显著降低(P < 0.05)。对于各组MSNs的组间比较可以发现, 50 nm组所产生的氧化应激水平显著高于其余各组(P < 0.01), 当浓度达到250 µg·mL-1时, 在巨噬细胞和内皮细胞中的SOD活性分别为1.34 ± 0.43和3.32 ± 0.38 U·mg-1 prot; 此时, 100 nm组的SOD活性与150、200 nm组也存在显著性差异(P < 0.05), 但对细胞的氧化应激增加程度不如50 nm组。结果表明, 随着浓度增加各组MSNs的SOD活性也呈下降趋势, 说明介孔二氧化硅诱导巨噬细胞和内皮细胞氧化应激的水平具有剂量相关性。
使用二氢乙啶(DHE) 活性氧ROS荧光探针对RAW264.7和HUVEC细胞进行染色, 通过荧光显微镜观察各粒径MSNs与细胞作用后, 观察细胞内产生荧光强弱的变化。图 4可见, MSNs能使细胞产生活性氧, 且两种细胞间的差异性不明显。从粒径的角度分析, 随着粒径增大, 红色荧光强度逐渐减弱; 从浓度的角度分析, 红色荧光强度随MSNs浓度升高显著增强, 说明MSNs诱导释放活性氧自由基具有明显的粒径依赖和浓度相关性, 此结果与SOD活性酶检测实验一致。
纳米颗粒能否从体内快速消除代谢是决定其毒性大小的关键。本研究拟通过体内分布、脏器中蓄积、药动学和尿液清除评价不同粒径MSNs在体内的消除。活体荧光和小鼠离体脏器荧光结果中(图 5A~C), 200 nm MSNs在比小粒径MSNs荧光下降更快, 而50~100 nm MSNs的荧光强峰值延续到12 h附近; 在注射72 h后, 小鼠离体脏器荧光结果显示MSNs在体内主要蓄积于肝脏, 肾脏次之, 少部分积留在肺部, 大粒径的MSNs肾脏荧光定量高于低粒径组, 因此推测大粒径MSNs消除方式可能是为肾脏途径排除。因此, 进一步考察了不同粒径的MSNs处理小鼠药代动力学过程和尿液排出量, 用于评价粒径与体内消除的相关性。图 5D显示了不同颗粒大小的不同MSNs在大鼠血液中的药代动力学过程。结果显示较小尺寸MSNs具有较长的血液循环周期, 注射1 h后血液药物浓度仍能达到约5 μg·mL-1 MSNs, 这可能是由于肝脏和脾脏等各种器官较慢捕获的结果[15]图 5E显示不同粒径MSNs处理后的Balb/c小鼠尿液在不同时间点的MSNs消除百分比, 反映体内不同时间段的平均降解率和降解产物的排泄量。在尾静脉注射后12 h内, MSNs降解产物从尿液排泄, 且排泄量随粒径大小显著增加, 这与荧光实验结果相似。基于结果, 具有较小粒径的50 nm MSNs具有最长的血液循环周期, 由于其在血液中的停留时间相对较长, 因此具有更多潜在的药物靶向和缓控释应用价值, 但是较大粒径MSNs似乎更能较快从体内降解和消除, 具有相对优异的体内安全性。综上所述, 粒径作为影响MSNs在体内降解消除的因素之一, 通过进一步研究筛选最佳粒径的MSNs能够达到最佳体内循环周期, 又能够快速消除, 这对加快拓展MSNs的临床运用具有重要意义。
纳米材料的设计和制造是一项重大的科学成就, 它影响着多个领域, 包括生物医学和制药领域[16-18]。安全评估是任何新技术或药品的一个组成部分, 纳米载体也不例外[19, 20]。为了探讨不同粒径MSNs的安全性, 通过体重监测、血常规和血清生化检测、主要组织病理学切片进行了体内安全性评价。给药后16天中, 各组小鼠体重变化如图 6A所示, 50 nm组相比于对照组略有下降趋势, 但各组小鼠体重变化并没有显著性差异, 证明MSNs具有较好的体内安全性, 且与粒径没有直接相关联系。血常规结果如图 6B所示, 各组与对照组未见显著性差异, 证明不同粒径(50~200 nm) MSNs都具有较好的生物相容性。本文还对主要组织(心、肝、脾、肺和肾脏) 的切片进行了HE染色, 用来进一步判断不同粒径MSNs对小鼠的脏器毒性。从图 6C可以看出, 150和200 nm组的肝脏和肾脏有极少部分的炎症细胞和坏死组织, 但整体主要组织没有明显损伤, 这与体重监测、血液检测结果相符。证明不同粒径(50~200 nm) MSNs具有良好的生物相容性, 且与粒径不呈相关联系, 当然也有可能是给药周期较短和粒径区间较小有关, 延长给药周期与放大粒径区间, 更能够考察不同粒径的体内安全性, 需要进一步相关实验验证。
本研究通过控制条件制备了不同粒径的MSNs (50、100、150、200 nm) 并在细胞、血液、器官/组织水平上考察了MSNs粒径与毒性的相关性。体外结果表明, MSNs对RAW264.7和HUMEC两种细胞的细胞毒性呈粒径相关性, 粒径越小其毒性越高, 在高浓度和更长孵育时间条件下, 趋势更加明显; 后续细胞共定位实验探讨其原因, 发现较小粒径MSNs的细胞内化程度高, 由于巨噬细胞RAW264.7对MSNs的摄取能力强于内皮细胞HUMEC, 其细胞毒性呈现相同趋势; 进一步发现较小粒径的MSNs更易诱导氧化应激, 造成超氧化物性损伤; 最后细胞凋亡实验再次验证了MSNs的细胞毒性具有粒径相关性; 溶血实验显示不同粒径MSNs具有相对较好的生物相容性, 且粒径因素对溶血毒性不造成影响。体内实验结果中, 活体荧光成像和离体组织荧光成像显示MSNs主要蓄积在小鼠肝脏与肾脏, 且大粒径MSNs在体内更易降解消除; 药动学和尿液药物检测结果显示, 不同粒径MSNs都主要经泌尿系统排泄, 且大粒径的MSNs较小粒径的MSNs更易降解排除; 经不同粒径MSNs处理后小鼠体重、血常规及主要脏器病理切片结果说明不同粒径MSNs具有良好的体内相容性, 对体重、血液及主要脏器不产生明显的毒性损伤影响。综上所述, 本研究制备粒度梯度的MSNs进行体内外毒性研究, 进一步考察不同粒径MSNs毒性与粒径的相关性, 为MSNs应用于递药系统的安全性设计提供新策略与新方法。
作者贡献: 谢晓伟负责文献查阅、实验设计、实验操作、数据分析、论文初稿撰写及根据编辑部修改意见进行修改; 程梦莹负责实验操作、样品采集和论文后续修改; 方威祥、林雪、顾文婷负责实验操作、文章图表格式处理; 余凯玲、叶婷娴、程苇钇和何莉参与论文修改; 李范珠、朴寄纲、郑杭生和魏颖慧参与理论指导、实验设计、论文修改及论文定稿。
利益冲突: 本文所有作者均同意发表, 所有的作者之间不存在利益冲突。
  • 国家自然科学基金面上项目(82074027)
  • 国家自然科学基金面上项目(82174096)
  • 国家自然科学基金青年项目(82104405)
  • 浙江省自然科学基金重点项目(LZ21H280001)
  • 浙江省自然科学基金探索项目(LY23H300002)
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2023年第58卷第8期
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doi: 10.16438/j.0513-4870.2023-0011
  • 接收时间:2023-01-05
  • 首发时间:2025-11-21
  • 出版时间:2023-08-12
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  • 收稿日期:2023-01-05
  • 修回日期:2023-05-13
基金
国家自然科学基金面上项目(82074027)
国家自然科学基金面上项目(82174096)
国家自然科学基金青年项目(82104405)
浙江省自然科学基金重点项目(LZ21H280001)
浙江省自然科学基金探索项目(LY23H300002)
作者信息
    浙江中医药大学药学院, 浙江 杭州 310000

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*朴寄纲, E-mail: ;
李范珠, Tel: 86-571-61768130, E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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