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Article(id=1218297483428545395, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218297478793843630, articleNumber=1001-2494(2024)14-1331-08, orderNo=null, doi=10.11669/cpj.2024.14.009, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1689091200000, receivedDateStr=2023-07-12, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768394354922, onlineDateStr=2026-01-14, pubDate=1721577600000, pubDateStr=2024-07-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768394354922, onlineIssueDateStr=2026-01-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768394354922, creator=13701087609, updateTime=1768394354922, updator=13701087609, issue=Issue{id=1218297478793843630, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='14', pageStart='1273', pageEnd='1358', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768394353817, creator=13701087609, updateTime=1768394585064, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1218298448764387533, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218297478793843630, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1218298448764387534, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218297478793843630, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1331, endPage=1338, ext={EN=ArticleExt(id=1218297483717952377, articleId=1218297483428545395, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Characterization and in Vitro Anticancer Ability of Doxorubicin Loaded Bimodal Mesoporous Silica Nanospheres, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To prepare bimodal mesoporous silica nanospheres loaded with doxorubicin, and evaluate their adsorption/ desorption properties and proliferation inhibition effects on cancer cells. METHODS bimodal mesoporous silica nanospheres(BMSNs) were prepared using sol-gel method and thermal treatment. The nanospheres were analyzed and characterized using scanning electron microscopy, specific surface area and porosity analyzer, Fourier transform infrared spectroscopy, and UV-VIS spectrometer. The adsorption/desorption characteristics of DOX by BMSNs were investigated by adsorption experiment. The proliferation inhibition effects of BMSNs-DOX on MCF-7 cells and A549 cells were detected using CCK8 method. RESULTS Compared with mesoporous MCM-41, which has typical uniform mesopores, BMSNs could provide more favorable matter diffusion and adsorption capacity towards DOX due to their more expansive pore structure. At pH=7.0, BMSNs exhibited a high adsorption capacity for DOX of 91.7 μmol·g-1 (equivalent to a drug loading capacity of 5.32%), which was 1.6 times higher than that of MCM-41. In pH=5.0 buffer solution, BMSNs-DOX desorbed (released) DOX molecules with an efficiency of about 96%, in just 30 minutes. BMSNs-DOX exhibited a stronger inhibitory effect than MCM-41-DOX and DOX. At a concentration of 4 μg·mL-1, the survival rate of MCF-7 cells and A549 cells was only 23.67% and 15.79% respectively. CONCLUSION Compared with MCM-41, BMSNs is expected to release DOX to cancer cells more efficiently. BMSNs-DOX exhibits a significant inhibitory effect on MCF-7 cells and A549 cells, suggesting its potential as a drug carrier to enhance the cytotoxicity of chemotherapy drugs on cancer cells.

, correspAuthors=Qingwei HU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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, authorCompany=null, fund=null, authors=null, authorsList=Xiaoya NIE, Xiaojuan ZHANG, Yan ZHANG, Shaobing GE, Qingwei HU), CN=ArticleExt(id=1218297486473610159, articleId=1218297483428545395, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=载多柔比星双介孔二氧化硅纳米球的表征及体外抗癌研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 制备载多柔比星(doxorubicin, DOX)的双介孔二氧化硅纳米球(bimodal mesoporous silica nanospheres, BMSNs),并评估其体外吸附/脱附特性和对肿瘤细胞增殖的抑制作用。方法 通过溶胶-凝胶法和溶剂热处理途径合成BMSNs;采用扫描电镜、比表面积及孔隙度分析仪、傅里叶变换红外光谱仪、紫外-可见光谱仪等方法对BMSNs进行分析和表征;采用间歇式吸附实验考察BMSNs对DOX的吸附/脱附特性;采用CCK8法检测载DOX双介孔二氧化硅纳米球BMSNs-DOX对人乳腺癌细胞MCF-7和人肺癌细胞A549增殖抑制作用。结果 BMSNs对DOX吸附/脱附能力明显优于典型均匀介孔二氧化硅MCM-41。在pH=7.0时,BMSNs对DOX的吸附量高达91.7 μmol·g-1(即载药量5.32%),是MCM-41吸附量的1.6倍。在pH=5.0缓冲溶液中,BMSNs-DOX对DOX分子的脱附(释放)效率可达约96%,脱附时间仅需约30 min。与MCM-41-DOX和DOX相比,BMSNs-DOX对人乳腺癌细胞MCF-7和人肺癌细胞A549增殖抑制作用更强,在4 μg·mL-1质量浓度时,癌细胞存活率仅为23.67%和15.79%。结论 相比于MCM-41,BMSNs能更快、更多地对癌症细胞释放抗癌药物DOX,BMSNs-DOX对 MCF-7 细胞和A549细胞有较强的杀伤作用,是一种具有潜在应用价值的新型给药系统。

, correspAuthors=胡清伟, authorNote=null, correspAuthorsNote=
* 胡清伟,男,本科,讲师 研究方向:肿瘤药理学 Tel:(023)691969199
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聂晓娅,女,本科,实验师 研究方向:新型药物传递系统

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聂晓娅,女,本科,实验师 研究方向:新型药物传递系统

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concentration(A) or pH condition(B). n=3,$\bar{x}±s$, figureFileSmall=S6a6+icXMN8le+o4GrX+bQ==, figureFileBig=CwSfJUY6RBIMJi9zCPgDUw==, tableContent=null), ArticleFig(id=1218297491028623440, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=CN, label=图3, caption=不同DOX初始浓度(A)、不同pH条件(B)下MCM-41和BMSNs对DOX的吸附性能。n=3,$\bar{x}±s$, figureFileSmall=S6a6+icXMN8le+o4GrX+bQ==, figureFileBig=CwSfJUY6RBIMJi9zCPgDUw==, tableContent=null), ArticleFig(id=1218297491095732307, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=EN, label=Fig.4, caption=Isothermal (A) and dynamic (B) adsorption model of BMSNs toward DOX (pH=7.0,T=30 ℃), figureFileSmall=k4yF9StoToJraTzHkaS2/Q==, figureFileBig=DwLNj97x+HO5R74CZXdTPA==, tableContent=null), ArticleFig(id=1218297491192201304, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=CN, label=图4, 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microsphere(C, D) on MCF-7 cells and A549 cells proliferation was evaluated by the CCK8 assay. n=6,$\bar{x}±s$

1)P<0.05,2)P<0.01, vs BMSNs-DOX group.

, figureFileSmall=NKmc8PgwBEwiBF1a+qHFtQ==, figureFileBig=rA+ltKlIFYd0m77ge2PKQA==, tableContent=null), ArticleFig(id=1218297491540328547, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=CN, label=图6, caption=CCK-8法检测不同浓度空白微球对MCF-7细胞(A)、A549细胞(B)细胞活力的影响;不同浓度载药微球对MCF-7细胞(C),A549细胞(D)细胞活力的影响。n=6,$\bar{x}±s$

与BMSNs-DOX组比,1)P<0.05,2)P<0.01。

, figureFileSmall=NKmc8PgwBEwiBF1a+qHFtQ==, figureFileBig=rA+ltKlIFYd0m77ge2PKQA==, tableContent=null), ArticleFig(id=1218297491615826022, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=EN, label=Tab.1, caption=

Isotherm parameters for adsorption models in the DOX adsorption on BMSNs

, figureFileSmall=null, figureFileBig=null, tableContent=
Adsorption model Formula Parameter Value
Freundlich qe=Kf C e 1 n f Kf/L·g-1 41.479
nf 2.607
r2 0.995 3
Redlich-Peterson qe= K r C e 1 + a r c e g Kr/L·g-1 0.543 04
αr -1.032 28
g 0.045 19
r2 0.753 2
Temkin qe= R T b Tln A T C e bT 381.066 8
AT 1.016 63
r2 0.970 7
), ArticleFig(id=1218297491682934890, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=CN, label=表1, caption=

BMSNs对DOX等温吸附模型拟合参数表

, figureFileSmall=null, figureFileBig=null, tableContent=
Adsorption model Formula Parameter Value
Freundlich qe=Kf C e 1 n f Kf/L·g-1 41.479
nf 2.607
r2 0.995 3
Redlich-Peterson qe= K r C e 1 + a r c e g Kr/L·g-1 0.543 04
αr -1.032 28
g 0.045 19
r2 0.753 2
Temkin qe= R T b Tln A T C e bT 381.066 8
AT 1.016 63
r2 0.970 7
), ArticleFig(id=1218297491758432365, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=EN, label=Tab.2, caption=

Kinetic parameters for kinetic models in the DOX adsorption on BMSNs

, figureFileSmall=null, figureFileBig=null, tableContent=
Kinetic models Formula Parameter Value
Pseudo-first model qt=qe 1 - e - K 1 t qe 91.717
K1/g·μmol-1·min-1 0.058 4
r2 0.942 33
Pseudo-second model qt= q e 2 K 2 t 1 + q e K 2 t qe 93.152
K2/g·μmol-1·min-1 0.001 29
r2 0.987 36
Elovich qt= 1 bln 1 + a b t α 70.249
b 0.075 18
r2 0.979 58
), ArticleFig(id=1218297491838124145, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218297483428545395, language=CN, label=表2, caption=

BMSNs对DOX吸附动力模型拟合参数表

, figureFileSmall=null, figureFileBig=null, tableContent=
Kinetic models Formula Parameter Value
Pseudo-first model qt=qe 1 - e - K 1 t qe 91.717
K1/g·μmol-1·min-1 0.058 4
r2 0.942 33
Pseudo-second model qt= q e 2 K 2 t 1 + q e K 2 t qe 93.152
K2/g·μmol-1·min-1 0.001 29
r2 0.987 36
Elovich qt= 1 bln 1 + a b t α 70.249
b 0.075 18
r2 0.979 58
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载多柔比星双介孔二氧化硅纳米球的表征及体外抗癌研究
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聂晓娅 1, 2 , 张小娟 1 , 张艳 1 , 葛少兵 3 , 胡清伟 1, 2, *
中国药学杂志 | 论著 2024,59(14): 1331-1338
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中国药学杂志 | 论著 2024, 59(14): 1331-1338
载多柔比星双介孔二氧化硅纳米球的表征及体外抗癌研究
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聂晓娅1, 2, 张小娟1, 张艳1, 葛少兵3, 胡清伟1, 2, *
作者信息
  • 1 重庆医药高等专科学校,重庆 401331
  • 2 重庆市药物制剂工程技术研究中心, 重庆 401331
  • 3 西北工业大学, 西安 710072

    • 聂晓娅,女,本科,实验师 研究方向:新型药物传递系统

通讯作者:

* 胡清伟,男,本科,讲师 研究方向:肿瘤药理学 Tel:(023)691969199
Characterization and in Vitro Anticancer Ability of Doxorubicin Loaded Bimodal Mesoporous Silica Nanospheres
Xiaoya NIE1, 2, Xiaojuan ZHANG1, Yan ZHANG1, Shaobing GE3, Qingwei HU1, 2, *
Affiliations
  • 1 Chongqing Medical and Pharmaceutical College, Chongqing 401331, China
  • 2 Chongqing Engineering Research Center of Pharmaceutical Sciences, Chongqing 401331, China
  • 3 Northwestern Polytechnical University, Xi'an 710072, China
出版时间: 2024-07-22 doi: 10.11669/cpj.2024.14.009
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摘要
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目的 制备载多柔比星(doxorubicin, DOX)的双介孔二氧化硅纳米球(bimodal mesoporous silica nanospheres, BMSNs),并评估其体外吸附/脱附特性和对肿瘤细胞增殖的抑制作用。方法 通过溶胶-凝胶法和溶剂热处理途径合成BMSNs;采用扫描电镜、比表面积及孔隙度分析仪、傅里叶变换红外光谱仪、紫外-可见光谱仪等方法对BMSNs进行分析和表征;采用间歇式吸附实验考察BMSNs对DOX的吸附/脱附特性;采用CCK8法检测载DOX双介孔二氧化硅纳米球BMSNs-DOX对人乳腺癌细胞MCF-7和人肺癌细胞A549增殖抑制作用。结果 BMSNs对DOX吸附/脱附能力明显优于典型均匀介孔二氧化硅MCM-41。在pH=7.0时,BMSNs对DOX的吸附量高达91.7 μmol·g-1(即载药量5.32%),是MCM-41吸附量的1.6倍。在pH=5.0缓冲溶液中,BMSNs-DOX对DOX分子的脱附(释放)效率可达约96%,脱附时间仅需约30 min。与MCM-41-DOX和DOX相比,BMSNs-DOX对人乳腺癌细胞MCF-7和人肺癌细胞A549增殖抑制作用更强,在4 μg·mL-1质量浓度时,癌细胞存活率仅为23.67%和15.79%。结论 相比于MCM-41,BMSNs能更快、更多地对癌症细胞释放抗癌药物DOX,BMSNs-DOX对 MCF-7 细胞和A549细胞有较强的杀伤作用,是一种具有潜在应用价值的新型给药系统。

双介孔二氧化硅纳米球  /  多柔比星  /  吸附  /  脱附  /  抗癌

OBJECTIVE To prepare bimodal mesoporous silica nanospheres loaded with doxorubicin, and evaluate their adsorption/ desorption properties and proliferation inhibition effects on cancer cells. METHODS bimodal mesoporous silica nanospheres(BMSNs) were prepared using sol-gel method and thermal treatment. The nanospheres were analyzed and characterized using scanning electron microscopy, specific surface area and porosity analyzer, Fourier transform infrared spectroscopy, and UV-VIS spectrometer. The adsorption/desorption characteristics of DOX by BMSNs were investigated by adsorption experiment. The proliferation inhibition effects of BMSNs-DOX on MCF-7 cells and A549 cells were detected using CCK8 method. RESULTS Compared with mesoporous MCM-41, which has typical uniform mesopores, BMSNs could provide more favorable matter diffusion and adsorption capacity towards DOX due to their more expansive pore structure. At pH=7.0, BMSNs exhibited a high adsorption capacity for DOX of 91.7 μmol·g-1 (equivalent to a drug loading capacity of 5.32%), which was 1.6 times higher than that of MCM-41. In pH=5.0 buffer solution, BMSNs-DOX desorbed (released) DOX molecules with an efficiency of about 96%, in just 30 minutes. BMSNs-DOX exhibited a stronger inhibitory effect than MCM-41-DOX and DOX. At a concentration of 4 μg·mL-1, the survival rate of MCF-7 cells and A549 cells was only 23.67% and 15.79% respectively. CONCLUSION Compared with MCM-41, BMSNs is expected to release DOX to cancer cells more efficiently. BMSNs-DOX exhibits a significant inhibitory effect on MCF-7 cells and A549 cells, suggesting its potential as a drug carrier to enhance the cytotoxicity of chemotherapy drugs on cancer cells.

bimodal mesoporous silica nanospheres  /  doxorubicin  /  adsorption  /  desorption  /  anticancer
聂晓娅, 张小娟, 张艳, 葛少兵, 胡清伟. 载多柔比星双介孔二氧化硅纳米球的表征及体外抗癌研究. 中国药学杂志, 2024 , 59 (14) : 1331 -1338 . DOI: 10.11669/cpj.2024.14.009
Xiaoya NIE, Xiaojuan ZHANG, Yan ZHANG, Shaobing GE, Qingwei HU. Characterization and in Vitro Anticancer Ability of Doxorubicin Loaded Bimodal Mesoporous Silica Nanospheres[J]. Chinese Pharmaceutical Journal, 2024 , 59 (14) : 1331 -1338 . DOI: 10.11669/cpj.2024.14.009
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随着空气、气候、饮食和生活方式等非遗传性因素的变化,人体内正常细胞发生基因突变、畸变甚至癌变的风险持续增高[1-4]。世界卫生组织国际癌症研究机构(IARC)发布2020年全球新发癌症病例1 929万例,癌症死亡病例996万例,给人类的生命健康和经济发展带来了严重的威胁[5-6]。目前用于癌症治疗的方法主要有手术疗法、放射疗法、免疫疗法和化学疗法等[7-8]。其中,化学疗法以某些药物能够干扰或杀死分裂中的癌细胞为理论基础,通过口服、静注等方式进行给药,并随着人体内部循环将药物扩散至各个器官,对处于全身性转移的癌症具有较好的疗效,已得到临床验证和应用[9-10]。但目前主流的化疗药物如多柔比星(doxorubicin,DOX)、紫杉醇、顺铂等,均被报道存在自身毒性、细胞摄取率低、副作用大等缺陷,大大限制了其临床应用[11-14]。因此采用负载量高、细胞摄取强、靶向特性好的药物载体和运输体系成为了改善化学治疗效果的有效途径之一[15-16]。
常见的药物载体材料有脂质、聚乙二醇和聚乳酸-羟基乙酸共聚物等。目前临床使用的有DOX脂质体,虽然其与DOX疗效相当,且毒副作用减少,但其粒径较大,稳定性相对较差,易被网状内皮系统识别和摄取,仍需进一步改进[17]。有研究表明非晶体或非粉尘状的介孔二氧化硅纳米颗粒具有良好的化学稳定性与生物兼容性[18];且其发达的孔道结构、大的孔容量和超高的比表面积,为大量药物分子的负载与运输提供了广阔空间[19-21];表面大量活跃的硅羟基(Si-OH)为多种形式的修饰与功能化奠定了坚实平台,这些均使得介孔二氧化硅在载药递送体系中展现出潜在的应用前景,被广泛用于抗癌药物负载、运输和释放等研究[18-24]。Roik等[25]通过溶胶-凝胶模板法合成的介孔二氧化硅MCM-41,其可通过静电和氢键作用对DOX产生显著的吸附能力。Gai等[26]采用自组装途径合成的聚磁性和介孔于一体的Fe3O4/二氧化硅微球,可将DOX运送到人子宫颈瘤细胞(HeLa)的细胞核,从而杀死癌细胞;对L929纤维细胞也具有高效的毒杀作用。基于上述研究背景,本实验以合成二维六方介孔氧化MCM-41为基础,通过溶胶-凝胶和溶剂热处理途径合成了具有孔径尺寸为2.6、4.1 nm的双介孔二氧化硅纳米球(BMSNs),成功制备了载DOX的BMSNs,并对其体外吸附/脱附特性和对肿瘤细胞增殖的抑制作用进行了考察。
1 仪器与试剂
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1.1 仪器
AUX220分析天平(日本Shimadzu公司);GL-3250B磁力搅拌器(其林贝尔仪器制造有限公司);Vortex-2旋涡混合器(上海沪析实业有限公司);VEGA3-LMH扫描电镜(捷克TESCAN有限公司);Tensor 27傅里叶变换红外光谱仪(德国BRUKER公司);BlueStar紫外-可见光谱仪(北京莱伯泰科仪器股份有限公司),TriStar II 3020全自动比表面积及孔隙度分析仪(美国Micromeritics仪器公司)。
1.2 试剂
盐酸DOX(大连美仑生物技术有限公司,纯度>98%,BR);十六烷基三甲基溴化铵(CTAB)、正硅酸乙酯(TEOS)、四甲基氢氧化铵(TMAOH)(成都市科隆化学品有限公司);氨水、无水乙醇、磷酸二氢钠、磷酸氢二钠(国药集团上海化学试剂公司);其他试剂均为分析纯,实验用水为超纯水。
2 方法
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2.1 介孔二氧化硅MCM-41的制备
在35℃持续搅拌条件下,将3.5 g CTAB完全溶于31.6 mL去离子水后,加入含有TEOS(17.1 mL)和TMAOH(8.1 mL)混合溶液,20 min后再缓慢加入15.4 mL TEOS溶液。1 h后将体系混合溶液转移至聚四氟乙烯反应釜中,在100 ℃下老化24 h。最后进行过滤、洗涤、干燥,并在500 ℃下煅烧6 h,反应结束后冷却至室温,用无水乙醇清洗5次,制得介孔二氧化硅MCM-41,冷冻干燥密封保存[27]。
2.2 BMSNs的制备
在35 ℃持续搅拌条件下,将3.0 g CTAB完全溶于61.5 mL去离子水后,加入17.5 mL氨水(质量分数25%~28%)和95.0 mL无水乙醇,15 min后逐滴加入5.3 mL TEOS溶液。2 h后对反应物进行过滤处理,并将所得固体产物分散于100 mL无水乙醇溶液后,转移至聚四氟乙烯反应釜,在100℃下进行溶剂热处理24 h。最后进行过滤、洗涤、干燥,并在500 ℃下煅烧6 h,反应结束后冷却至室温,用无水乙醇清洗5次,制得双介孔二氧化硅纳米球BMSNs,冷冻干燥密封保存,制备过程见图1。
2.3 分析与表征
样品形貌通过VEGA3-LMH扫描电镜(SEM)观测。氮气吸附/脱附等温曲线通过TriStar Ⅱ 3020仪器测得,测试前样品需预先真空脱气处理(80 ℃/12 h),材料比表面积、孔径分布分别通过Brunauer-Emmet-Teller、Barrett-Joyner-Halenda方法计算得到。傅立叶红外光谱(FTIR)由Bruker Tensor 27型傅立叶红外光谱仪进行表征,波数范围400~4 000 cm-1,分辨率为4 cm-1。DOX浓度通过BlueStar系列紫外-可见光谱测定(λmax=480 nm)。
2.4 吸附特性实验
吸附性能测试实验均通过间歇式途径完成,详细操作过程为:20 mg吸附剂材料与20 mL预定浓度的DOX溶液混合,在30 ℃条件下振荡24 h之后,通过滤膜孔径为0.22 μm的过滤器进行过滤,溶液中DOX浓度通过紫外-可见分光光度计测定。平衡吸附量qe(μmol·g-1)由公式1计算得到:
qe= c o - c e m× V 1   000
式中:qe(μmol·g-1)为样品的平衡吸附量,m(g)为样品的质量,V(mL)为吸附质溶液的体积,co(μmol·L-1)和ce(μmol·L-1)分别为溶液的初始摩尔浓度和吸附平衡后溶液的摩尔浓度。
pH值影响试验:取6只清洁的100 mL锥形瓶,分别加入20 mg吸附剂(BMSNs、MCM-41)和浓度为100 μmol·L-1 DOX溶液,溶液pH通过pH缓冲液依次调整为3.0、3.8、5.0、6.0、6.8、8.0,体积为20 mL。在30 ℃下,以50 r·min-1恒温振荡24 h后,对溶液进行过滤,并测定溶液中DOX浓度ce。按照公式1计算BMSNs、MCM-41在不同pH条件下的平衡吸附量qe。
等温吸附实验:取7只清洁的100 mL锥形瓶,分别加入20 mg BMSNs和20 mL初始浓度不同DOX溶液(70、100、135、160、190、220、250 μmol·L-1)。在30 ℃下,以50 r·min-1恒温振荡24 h后,对溶液进行过滤,并测定溶液中DOX浓度ce。按照公式1计算BMSNs在不同初始浓度条件下的平衡吸附量qe,得到对应等温吸附曲线。
吸附动力学实验:取7只清洁的100 mL锥形瓶,分别加入20 mg BMSNs和20 mL初始浓度为100 μmol·L-1的DOX溶液。在30 ℃以下以50 r·min-1恒温振荡,对应吸附时间的依次为5、20、40、60、120、200、360 min后,对溶液进行过滤,并测定溶液中DOX浓度(ct),由公式2得出BMSNs在不同吸附时间内对DOX的吸附量qt,并得到对应的吸附动力学曲线。
qt= c o - c t m× V 1   000
式中:qt(μmol·g-1)为样品在不同反应时间内对DOX的吸附量,ct(μmol·L-1)为样品与溶液接触t min后DOX的摩尔浓度。
脱附(释放)实验:取2只清洁的100 mL锥形瓶,加入20 mg在100 μmol·L-1的DOX溶液(pH=7.0)中吸附完成的BMSNs干燥材料,分别加入pH为5.0或7.4的缓冲溶液(20 mL)。在30 ℃下以50 r·min-1恒温振荡,在一定时间间隔内进行取样、过滤,并测定计算溶液中DOX浓度及含量m1,由公式3得出BMSNs在不同pH下对DOX的脱附(释放)效率η(%)。
η(%)= m 1 m×100%
式中:m(μmol)为20 mg BMSNs干燥材料在100 μmol·L-1的DOX溶液(pH=7.0)中的吸附量,m1(μmol)为缓冲溶液中DOX含量。
2.5 空白纳米球的毒性实验
评估空白纳米球MCM-41和BMSNs的细胞毒性,排出药物载体毒性的干扰。采用CCK-8法测定MCM-41和BMSNs对人乳腺癌细胞MCF-7、人肺癌细胞A549增殖活力的影响。取对数生长期的MCF-7细胞、A549细胞接种于96孔板中,细胞密度为每孔4×103个,在37 ℃、5% CO2的细胞培养箱中孵育过夜。次日将培养基更换为200 μL含不同质量浓度(10、25、50、100 μg·mL-1) MCM-41和BMSNs的新鲜培养基,同时设置调零组、对照组,各个浓度平行做6个复孔。培养48 h后,每孔加入20 μL CCK-8溶液,继续孵育1 h后,用酶标仪检测各孔在450 nm波长的吸光度(A)值,按公式4计算细胞存活率:
细胞存活率 %= A 实 验 组 - A 调 零 组 A 对 照 组 - A 调 零 组×100%
2.6 载药纳米球抑制肿瘤增殖作用实验
采用CCK-8法测定载药纳米球BMSNs-DOX和MCM-41-DOX对人乳腺癌细胞MCF-7、人肺癌细胞A549的增殖抑制作用。按上述方法将人乳腺癌细胞MCF-7、人肺癌细胞A549接种在96孔板并孵育过夜。次日,将培养基更换为200 μL含系列质量浓度(0.1、0.5、1.0、2.0、4.0、8.0 μg·mL-1)DOX、MCM-41-DOX、BMSNs-DOX的培养基,同时设置调零组、对照组,各6个复孔。培养48 h后,每孔加入20 μL CCK-8溶液,继续孵育1 h后,用酶标仪检测各孔在450 nm波长的吸收值,计算细胞存活率,方法同上。
数据处理 所有实验平行操作3次,数据采用SPSS 22.0软件统计分析,数据采用平均值±标准差(Mean±SD)表示,组间比较采用t检验,P<0.05视为差异具有统计学意义。
3 结果与分析
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3.1 结构表征与分析
为确认MCM-41和BMSNs样品形貌,采用SEM进行表征,结果见图2A~B。可见,MCM-41纳米颗粒形貌为类球形,但颗粒间交联现象明显,颗粒粒径约为520~960 nm;BMSNs为球形颗粒,分散性明显优于MCM-41,粒径约为420~780 nm。为进一步表征其孔结构,在-196 ℃条件下对MCM-41和BMSNs样品进行了氮气吸附脱附测试,结果见图2C。根据IUPAC定义可知[28],MCM-41和BMSNs氮气吸附脱附等温曲线均属于Ⅳ类曲线,MCM-41滞后环为H1型,BMSNs滞后环为H2型,说明两者都具有较发达的介孔结构。MCM-41和BMSNs比表面积分别为967.2、1 043.6 m2·g-1;孔体积容量分别为0.68、0.91 cm3·g-1。图2D表明MCM-41的孔径分布均匀,约为2.4 nm;BMSNs具有孔径约为2.6、4.1 nm的双介孔。图2E显示MCM-41和BMSNs均出现了属于SiO2物质的FTIR吸收谱图,无其他杂峰,说明在洗涤、模板去除等样品过程中进行彻底,样品较纯。其中位于3 450 cm-1吸收峰由O-H键伸缩振动引起,1 650 cm-1吸收峰来源于Si-OH和吸附H2O的弯曲振动,960 cm-1吸收峰为Si-OH对称伸缩振动,1 080、800、457 cm-1吸收峰分别归因于Si-O-Si键的不对称伸缩振动、对称伸缩振动和弯曲振动。
3.2 吸附特性研究
为研究BMSNs和MCM-41对抗癌药物分子DOX的吸附能力,分别考察了两者在不同DOX溶液和不同环境pH条件下对DOX的吸附平衡能力,结果见图3。由图3A可以发现,BMSNs和MCM-41对DOX的吸附能力均随着初始浓度的增大而增大,这是由于溶液中吸附质分子的浓度增加,使DOX具有更强的物质传输动力和更好的物质扩散能力,使其更容易被BMSNs和MCM-41吸附。
随着溶液pH值由3.0调整至8.0,BMSNs和MCM-41对DOX均呈现出先增加后降低的吸附能力(图3B),说明pH均对BMSNs和MCM-41的吸附能力具有显著影响。这是因为不同pH环境对DOX在溶液中的分子电荷状态以及SiO2表面Si-OH的电离程度均具有较大影响[27]。在酸性条件下,DOX大量被质子化呈现阳离子状态(图2F),SiO2表面以Si-OH形式存在,SiO2对DOX的吸附主要通过范德华力。在碱性条件下,DOX主要以非离子状态存在,SiO2表面Si-OH电离为Si-O-阴离子,SiO2对DOX的吸附也主要通过范德华力。因此BMSNs和MCM-41在酸、碱条件下均对DOX产生相对较低的吸附能力。而当溶液呈中性时,部分DOX分子被质子化呈阳离子状态,SiO2表面部分Si-OH电离为电负性的Si-O-阴离子,SiO2可通过静电作用对DOX进行吸附;且未质子化的DOX可与表面Si-OH通过氢键作用进行吸附,所以pH=7.0时,BMSNs和MCM-41对DOX的吸附能力显著增加。
另一方面,在相同的DOX初始浓度(图3A)、pH条件(图3B)下, BMSNs(孔径尺寸2.6、4.1 nm)对DOX的吸附能力均明显优于MCM-41(孔径尺寸2.4 nm)。特别是在pH=7.0时,BMSNs对DOX的吸附量高达91.7 μmol·g-1(即载药量为5.32%),是MCM-41吸附量的1.6倍。说明具有2种不同小大孔径的BMSNs,更有利于DOX分子在其内外表面的扩散和运输,能产生更优异的吸附能力,可作为DOX高负载量载体。
为进一步探究BMSNs对DOX的吸附特性,对其等温吸附、动力学吸附进行了实验和理论拟合。同时根据肿瘤组织中pH值比正常细胞更低的特点,尤其是在癌细胞内pH=6.5~6.8,溶酶体内pH=4.5~6.5 [29-31]。对其在不同pH条件下的脱附(释放)性质进行了验证。由图4A可知,BMSNs对DOX的吸附平衡量随着平衡浓度的增加而呈现出非线性式增加趋势。通过3种典型的吸附模型(Freundlich、Redlich-Peterso和Temkin)对等温吸附数据进行了拟合,由表1可知,该过程最符Freundlich模型(r2=0.995 3,Kf=41.479 L·g-1)。BMSNs主要通过不均匀的作用力(静电吸附和氢键作用)对DOX产生吸附。由图4B可知,BMSNs对DOX的吸附速率较快,在DOX初始浓度为100 μmol·L-1时,BMSNs在约120 min内可实现对DOX的饱和吸附。通过3种典型的动力学模型(准一级动力学、准二级动力学和Elovich模型)对动力学实验数据进行了拟合分析,由表2可知,该过程最符合于准二级动力学吸附模型(r2=0.987 36,K2=0.001 29 g·μmol-1·min-1)。由图5可知,在pH=7.0条件下吸附完成的BMSNs-DOX,均可在pH=5.0和pH=7.4的缓冲溶液中进行DOX分子脱附/释放。且在pH=5.0溶液中,BMSNs对DOX分子的脱附(释放)效率可达约96%,脱附时间仅需约30 min,脱附(释放)速率和效率均显著优于在pH=7.4环境中的DOX脱附/释放,说明BMSNs可实现对癌症细胞释放更多更快的抗癌药物分子DOX,增强对癌细胞的破坏和杀死作用;同时减少在正常细胞内释放DOX,降低副作用,在作为癌细胞特异性药物载体方面具有潜在应用价值。
3.3 体外药效学评价
为了评价空白载体的毒性,分别将MCM-41和BMSNs加入 MCF-7和A549细胞中,共同孵育48 h 后,采用 CCK8法测定细胞的存活率。实验结果见图6A~B,与2种空白纳米球共孵育的肿瘤细胞存活率均高于90%,表明空白MCM-41和BMSNs毒性小,生物相容性好,是安全的药物载体。为了验证载药纳米球BMSNs-DOX可以增强DOX的抗肿瘤效果,将不同浓度的DOX、MCM-41-DOX、BMSNs-DOX分别加入到MCF-7和A549细胞中,48 h后采用CCK8法测定肿瘤细胞存活率。实验结果见图6C~D,随着DOX浓度的增加,肿瘤细胞存活率逐渐降低,在相同药物浓度下,BMSNs-DOX组相较于DOX组和MCM-41-DOX组,展现出更强的肿瘤细胞杀伤能力。在DOX 4 μg·mL-1质量浓度下,BMSNs-DOX组MCF-7和A549细胞存活率仅为23.67%和15.79%,在DOX 8 μg·mL-1质量浓度下,BMSNs-DOX组MCF-7和A549细胞存活率仅为21.85%和6.81%。
4 结论
收起
本研究以CTAB为结构导向剂,TEOS为硅源,在碱性条件下进行溶胶-凝胶合成后,通过溶剂热处理途径合成了双介孔二氧化硅纳米球,孔径尺寸为2.6、4.1 nm,比表面积高达1 043.6 m2·g-1。通过吸附实验表明:①BMSNs对DOX的吸附能力均明显优于具有典型介孔的MCM-41(孔径尺寸为2.4 nm),特别是在pH=7.0条件下,BMSNs对DOX的吸附量高达91.7 μmol·g-1(co=100 μmol·L-1),是MCM-41吸附量的1.6倍;②BMSNs主要通过静电作用和氢键作用对DOX产生优异吸附作用,属于Freundlich等温吸附模型、准二级动力学吸附;③在pH=5.0缓冲溶液中,BMSNs对DOX分子的脱附(释放)效率可达约96%,脱附时间仅需约30 min,明显优于其在pH=7.4溶液中的脱附(释放)量和速率。BMSNs-DOX纳米球给药后可先通过EPR效应富集到肿瘤组织,然后被内吞进入肿瘤细胞溶酶体释放药物,药物逃逸溶酶体进入细胞核发挥药效。以上研究表明BMSNs有望实现对癌症细胞释放更多、更快的抗癌药物DOX以增强杀死癌细胞作用,提高药效,减少毒副作用。同时本研究在体外药效学实验中观察到BMSNs-DOX组相较于DOX组和MCM-41-DOX组表现出更好的肿瘤细胞杀伤作用。综上所述,表明BMSNs是一种较理想的癌细胞特异性递药载体,具有潜在的应用价值。
基金
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  • 重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0627)
  • 重庆市教委科学技术研究青年项目(KJQN201902803)
  • 重庆医药高等专科学校科研教研项目(CQYGZJG2003)
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2024年第59卷第14期
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doi: 10.11669/cpj.2024.14.009
  • 接收时间：2023-07-12
  • 首发时间：2026-01-14
  • 出版时间：2024-07-22
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  • 收稿日期：2023-07-12
基金
重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0627)
重庆市教委科学技术研究青年项目(KJQN201902803)
重庆医药高等专科学校科研教研项目(CQYGZJG2003)
作者信息
    1 重庆医药高等专科学校,重庆 401331
    2 重庆市药物制剂工程技术研究中心, 重庆 401331
    3 西北工业大学, 西安 710072

通讯作者:

* 胡清伟,男,本科,讲师 研究方向:肿瘤药理学 Tel:(023)691969199
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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