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Article(id=1198628611759632843, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198628599835227075, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-1264, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1669219200000, receivedDateStr=2022-11-24, revisedDate=1671897600000, revisedDateStr=2022-12-25, acceptedDate=null, acceptedDateStr=null, onlineDate=1763704930486, onlineDateStr=2025-11-21, pubDate=1686499200000, pubDateStr=2023-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763704930486, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763704930486, creator=13701087609, updateTime=1763704930486, updator=13701087609, issue=Issue{id=1198628599835227075, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='6', pageStart='0', pageEnd='1724', issueExtLink='null', onlineDate='null', pubDate='1686499200000', pubDateStr='2023-06-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763704927643, creator='13701087609', updateTime=1766137690373, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1208832346359468483, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198628599835227075, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208832346359468484, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198628599835227075, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1669, endPage=1676, ext={EN=ArticleExt(id=1198628612200034793, articleId=1198628611759632843, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Construction and preliminary application of drug carrier system based on yeast membrane vesicles, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

As an edible eukaryotic microorganism, Saccharomyces cerevisiae has the characteristics of high safety, rapid proliferation, low cost, easy transformation, etc. It has been widely used to produce vaccines, antibodies, insulin, etc. Up to now, yeast components, such as cell wall and yeast microcapsules, have been widely used in the treatment of tumors, inflammatory virus infection, post-traumatic osteoarthritis and other diseases. Among them, the components of yeast cell membrane are relatively simple and stable, which are easy to be extracted on a large scale. Therefore, yeast cell membrane material was used to construct yeast membrane vesicle nanosystem, and its biomedical application was preliminarily explored. In this study, Saccharomyces cerevisiae membrane vesicle (SMV) was prepared by co-extrusion method, and the particle size and surface potential of SMV, drug loading and release characteristics, stability, cell safety, and in vitro therapeutic effect were investigated. The results showed that the average particle size of SMV was 185.1 nm. Curcumin and silica nanoparticles were effectively encapsulated by co-incubation and ultrasonic methods, and the characteristics of cell membrane proteins were maintained. Moreover, SMV had good stability and biocompatibility. In addition, SMV could be effectively uptaken by macrophages RAW 264.7, and curcumin loaded SMV could effectively eliminate reactive oxygen species (ROS). In conclusion, the yeast plasma membrane vesicles prepared in this study could effectively deliver curcumin drugs and encapsulate nanoparticles, and could be effectively absorbed by macrophages and effectively eliminate ROS, providing new ideas and new methods for biomedical applications of yeast membrane materials.

, authors=null, authorsList=Peng-cheng ZHAO, Ying-ying ZHANG, Ming-hui ZHANG, Han-jie WANG, authorCompany=null, correspAuthors=Han-jie WANG, 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=1198628613777093262, articleId=1198628611759632843, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于酵母膜囊泡药物载体系统的构建与应用初探, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

酿酒酵母作为可食用真核微生物, 具有安全性高、增殖快、成本低、易改造等特性, 已广泛用于生产疫苗、抗体、胰岛素等。截至目前, 酵母相关组分如细胞壁、酵母微囊体已被广泛用于肿瘤、炎症病毒感染、创伤后骨关节炎等疾病的治疗。其中, 酵母细胞膜组成成分相对简单稳定, 易于大规模提取。因此, 本研究以酵母细胞膜材料为研究对象, 构建酵母膜囊泡纳米系统, 并初步探究其生物医学应用。通过共挤出法制备酵母质膜囊泡(Saccharomyces cerevisiae membrane vesicle, SMV), 并对SMV的粒径与表面电位、药物装载与释放特性、稳定性、细胞安全性、体外治疗效果等进行探究。结果显示: SMV平均粒径为185.1 nm, 通过共孵育及超声等方法有效包载姜黄素及二氧化硅纳米粒, 并保持细胞膜蛋白特性。而且SMV具有良好的稳定性与生物相容性。此外, SMV可被巨噬细胞RAW 264.7有效摄取, 包载姜黄素的SMV可有效清除巨噬细胞内的活性氧(ROS)。综上, 本研究所制备的酵母质膜囊泡可有效递送姜黄素药物及包载纳米粒, 有效被巨噬细胞摄取并降低ROS, 为酵母膜材料的生物医学应用提供了新思路和新方法。

, authors=null, authorsList=赵鹏程, 张英英, 张明慧, 王汉杰, authorCompany=null, correspAuthors=王汉杰, authorNote=null, correspAuthorsNote=
*王汉杰, Tel: 15503391895, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2023, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=bMff5qsdBbQp9aEKol/AIQ==, magXml=zexpkBvdxo6KHs+hzcm/VQ==, pdfUrl=null, pdf=hj76Pb5Xx/7At16KmjhSzQ==, pdfFileSize=5411796, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=z46NbIgwhiav/2cZpXLzew==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=FoX7OY2n0mFjcZpD9MQYsg==, mapNumber=null, fund=null)}, authors=[Author(id=1198960134614515750, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1198960134803259452, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, authorId=1198960134614515750, language=EN, stringName=Peng-cheng ZHAO, firstName=Peng-cheng, middleName=null, lastName=ZHAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, address=1. 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Synth Biol J (合成生物学), 2022, 3: 810-820., articleTitle=Coated probiotic-based drug carriers for oral delivery of tumor antigens, refAbstract=null)], funds=[Fund(id=1198960140192940646, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, awardId=2019YFA0906500, language=CN, fundingSource=国家重点研发计划项目(2019YFA0906500), fundOrder=null, country=null), Fund(id=1198960140352324211, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, awardId=32122047, language=CN, fundingSource=国家自然科学基金优秀青年科学基金项目(32122047), fundOrder=null, country=null), Fund(id=1198960140515902089, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, awardId=31971300, language=CN, fundingSource=国家自然科学基金资助项目(31971300), fundOrder=null, country=null), Fund(id=1198960140612371090, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, awardId=81771970, language=CN, fundingSource=国家自然科学基金资助项目(81771970), fundOrder=null, country=null), Fund(id=1198960140746588832, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, awardId=19YFZCSY00190, language=CN, fundingSource=天津市重点研究与开发计划项目(19YFZCSY00190), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1198960134195086317, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, xref=null, ext=[AuthorCompanyExt(id=1198960134211863538, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, companyId=1198960134195086317, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. School of Life Sciences, Tianjin University, Tianjin 300072, China), AuthorCompanyExt(id=1198960134237029364, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, companyId=1198960134195086317, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.天津大学生命科学学院, 天津 300072)]), AuthorCompany(id=1198960134408994821, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, xref=null, ext=[AuthorCompanyExt(id=1198960134421577735, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, companyId=1198960134408994821, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2. School of Medical Imaging, Xuzhou Medical University, Xuzhou 221004, China), AuthorCompanyExt(id=1198960134429966345, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, companyId=1198960134408994821, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.徐州医科大学医学影像学院, 江苏 徐州 221004)])], figs=[ArticleFig(id=1198960138112565648, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=hTPZMEPShmEg3aHCiLka0g==, figureFileBig=LOWDz7D2HGaMTVxnobiaxw==, tableContent=null), ArticleFig(id=1198960138204840348, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 1, caption= Characterizations of yeast plasma membrane. A: Schematic diagram of yeast plasma membrane extraction; B: The changes of absorbance at 600 nm with time when yeast cells were pretreated and enzymatically hydrolyzed by snail enzyme. <i>n</i> = 3, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i>; C: The changes of absorbance at 600 nm with time when yeast cells of different concentrations were hydrolyzed by snail enzyme. <i>n</i> = 3, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i>; D: Observation of yeast cells and protoplasts under light microscope. Scale bar: 50 μm; E: Confocal imaging of Hoechst and DiI stained protoplasts and yeast cells. Scale bar: 100 μm; F: Sucrose gradient centrifugation of yeast cell breaking liquid; G: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of cell breaking liquid (1) and yeast plasma membrane (2) , figureFileSmall=hTPZMEPShmEg3aHCiLka0g==, figureFileBig=LOWDz7D2HGaMTVxnobiaxw==, tableContent=null), ArticleFig(id=1198960138381001129, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=apZZ8T0xG9EdERS3tKnW6Q==, figureFileBig=4uO/NbxQqlT8YQ+EkObrqg==, tableContent=null), ArticleFig(id=1198960138536190389, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 2, caption= Characterizations of <i>Saccharomyces cerevisiae</i> membrane vesicle (SMV). A: Transmission electron microscope (TEM) image of SMV. Scale bar: 100 nm; B: Particle size of SMV; C: The stability of SMV with time. PBS: Phosphate buffered saline; SIF: Simulated intestinal fluid; SGF: Simulated gastric fluid , figureFileSmall=apZZ8T0xG9EdERS3tKnW6Q==, figureFileBig=4uO/NbxQqlT8YQ+EkObrqg==, tableContent=null), ArticleFig(id=1198960138691379659, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=x+8jiEzCoao6xyX/tQolgg==, figureFileBig=sD9SQ7IWCaafMl2D8eHMMg==, tableContent=null), ArticleFig(id=1198960138901094888, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 3, caption= Cytotoxicity of SMV. A: After incubation with different concentrations of SMV for 24 h, living cells and dead cells double stained with calcein AM/PI living/dead cell double staining kit were observed under confocal microscope. Scale bar: 100 μm; B: The average PI fluorescence intensity in different groups; C: Cell viability of Caco-2 macrophages exposed to SMV for 24 h. <i>n</i> = 6, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i>; D: Cell viability of RAW 264.7 macrophages exposed to SMV for 12, 24 and 48 h. <i>n</i> = 6, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i> , figureFileSmall=x+8jiEzCoao6xyX/tQolgg==, figureFileBig=sD9SQ7IWCaafMl2D8eHMMg==, tableContent=null), ArticleFig(id=1198960139081449975, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=M9n3MhM8Fnlm+dNOsMg/MQ==, figureFileBig=zFISCia/Ry1Ei6uxHZsMSA==, tableContent=null), ArticleFig(id=1198960139211473417, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 4, caption= Cellular uptake of SMV. A: Confocal imaging of SMV taken by RAW 264.7 macrophages. Scale bar: 5 μm; B: Confocal images of SMV gradually taken by RAW 264.7 macrophages over time. Scale bar: 20 μm , figureFileSmall=M9n3MhM8Fnlm+dNOsMg/MQ==, figureFileBig=zFISCia/Ry1Ei6uxHZsMSA==, tableContent=null), ArticleFig(id=1198960139358274074, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=UGrl82LxyQT5DNkNesEadg==, figureFileBig=sDacrv2aZValJrmhGK8Png==, tableContent=null), ArticleFig(id=1198960139450548777, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 5, caption= Characterizations of SMV@SiO<sub>2</sub>. A: TEM images of SiO<sub>2</sub> and SMV@SiO<sub>2</sub>. Scale bar: 100 μm; B: Particle size distributions of SiO<sub>2</sub> and SMV@SiO<sub>2</sub>; C, D: Protein content analysis (C) and SDS-PAGE (D) of SMV, SiO<sub>2</sub> and SMV@SiO<sub>2</sub>; E: The stability of SMV@SiO<sub>2</sub> , figureFileSmall=UGrl82LxyQT5DNkNesEadg==, figureFileBig=sDacrv2aZValJrmhGK8Png==, tableContent=null), ArticleFig(id=1198960139567989299, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=EN, label=null, caption=null, figureFileSmall=/iL4MJFsT07+MNvOGpNWZg==, figureFileBig=m104zV4l34l5tPprkjNDfQ==, tableContent=null), ArticleFig(id=1198960139752538695, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628611759632843, language=CN, label=Figure 6, caption= Characterizations of SMV-curcumin (Cur). A: Ultraviolet and visible spectrum absorption spectra of SMV, Cur and SMV-Cur; B: Drug loading rate by different drug loading methods (co-incubation, repeated freezing and thawing, ultrasound). <i>n</i> = 3, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i>; C: Drug release behavior of SMV-Cur. <i>n</i> = 3, <span class="mag-xml-overline" style="border-top:1px solid black"><i>x</i></span> ± <i>s</i>; D: <i>In vitro</i> anti-inflammatory effects of SMV-Cur verified by reactive oxygen species probe (DCFH-DA). 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基于酵母膜囊泡药物载体系统的构建与应用初探
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赵鹏程 1 , 张英英 2 , 张明慧 1 , 王汉杰 1, *
药学学报 | 研究论文 2023,58(6): 1669-1676
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药学学报 |研究论文 2023 , 58 (6) : 1669 -1676
基于酵母膜囊泡药物载体系统的构建与应用初探
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赵鹏程1, 张英英2, 张明慧1, 王汉杰1, *
作者信息
  • 1.天津大学生命科学学院, 天津 300072
  • 2.徐州医科大学医学影像学院, 江苏 徐州 221004
通讯作者:
*王汉杰, Tel: 15503391895, E-mail:
Construction and preliminary application of drug carrier system based on yeast membrane vesicles
Peng-cheng ZHAO1, Ying-ying ZHANG2, Ming-hui ZHANG1, Han-jie WANG1, *
Affiliations
  • 1. School of Life Sciences, Tianjin University, Tianjin 300072, China
  • 2. School of Medical Imaging, Xuzhou Medical University, Xuzhou 221004, China
出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1264
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摘要
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酿酒酵母作为可食用真核微生物, 具有安全性高、增殖快、成本低、易改造等特性, 已广泛用于生产疫苗、抗体、胰岛素等。截至目前, 酵母相关组分如细胞壁、酵母微囊体已被广泛用于肿瘤、炎症病毒感染、创伤后骨关节炎等疾病的治疗。其中, 酵母细胞膜组成成分相对简单稳定, 易于大规模提取。因此, 本研究以酵母细胞膜材料为研究对象, 构建酵母膜囊泡纳米系统, 并初步探究其生物医学应用。通过共挤出法制备酵母质膜囊泡(Saccharomyces cerevisiae membrane vesicle, SMV), 并对SMV的粒径与表面电位、药物装载与释放特性、稳定性、细胞安全性、体外治疗效果等进行探究。结果显示: SMV平均粒径为185.1 nm, 通过共孵育及超声等方法有效包载姜黄素及二氧化硅纳米粒, 并保持细胞膜蛋白特性。而且SMV具有良好的稳定性与生物相容性。此外, SMV可被巨噬细胞RAW 264.7有效摄取, 包载姜黄素的SMV可有效清除巨噬细胞内的活性氧(ROS)。综上, 本研究所制备的酵母质膜囊泡可有效递送姜黄素药物及包载纳米粒, 有效被巨噬细胞摄取并降低ROS, 为酵母膜材料的生物医学应用提供了新思路和新方法。

细胞衍生的颗粒  /  纳米药物  /  酿酒酵母  /  细胞膜  /  纳米载药系统

As an edible eukaryotic microorganism, Saccharomyces cerevisiae has the characteristics of high safety, rapid proliferation, low cost, easy transformation, etc. It has been widely used to produce vaccines, antibodies, insulin, etc. Up to now, yeast components, such as cell wall and yeast microcapsules, have been widely used in the treatment of tumors, inflammatory virus infection, post-traumatic osteoarthritis and other diseases. Among them, the components of yeast cell membrane are relatively simple and stable, which are easy to be extracted on a large scale. Therefore, yeast cell membrane material was used to construct yeast membrane vesicle nanosystem, and its biomedical application was preliminarily explored. In this study, Saccharomyces cerevisiae membrane vesicle (SMV) was prepared by co-extrusion method, and the particle size and surface potential of SMV, drug loading and release characteristics, stability, cell safety, and in vitro therapeutic effect were investigated. The results showed that the average particle size of SMV was 185.1 nm. Curcumin and silica nanoparticles were effectively encapsulated by co-incubation and ultrasonic methods, and the characteristics of cell membrane proteins were maintained. Moreover, SMV had good stability and biocompatibility. In addition, SMV could be effectively uptaken by macrophages RAW 264.7, and curcumin loaded SMV could effectively eliminate reactive oxygen species (ROS). In conclusion, the yeast plasma membrane vesicles prepared in this study could effectively deliver curcumin drugs and encapsulate nanoparticles, and could be effectively absorbed by macrophages and effectively eliminate ROS, providing new ideas and new methods for biomedical applications of yeast membrane materials.

cell-derived microparticle  /  nanomedicine  /  Saccharomyces cerevisiae  /  cell membrane  /  nanoparticle drug delivery system
赵鹏程, 张英英, 张明慧, 王汉杰. 基于酵母膜囊泡药物载体系统的构建与应用初探. 药学学报, 2023 , 58 (6) : 1669 -1676 . DOI: 10.16438/j.0513-4870.2022-1264
Peng-cheng ZHAO, Ying-ying ZHANG, Ming-hui ZHANG, Han-jie WANG. Construction and preliminary application of drug carrier system based on yeast membrane vesicles[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1669 -1676 . DOI: 10.16438/j.0513-4870.2022-1264
正文
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酵母是一类异质真核真菌, 主要以单细胞形式存在。它具有许多优良特性, 如高分泌能力、高生长速率、蛋白翻译后修饰、易于培养、产物易于纯化等, 这些优良特性使其在工业、生物技术、医学和环境领域都有广泛应用[1-3]。例如, 酵母细胞可用于天然酶、生物乙醇、脂肪酸等活性物质的生产[4-6]。此外, 酵母作为食品中可食用的部分, 具有很高的安全性。国内外科学家利用酵母来打造多功能益生菌, 用于炎症性肠炎的治疗[7-10]。
酵母细胞具有安全无毒、无致病性、培养条件简单、易于基因改造等特性, 已被广泛用于生产疫苗、抗体、胰岛素等[11-14]。此外, 酵母细胞的结构及营养价值使其成为食品和药品工业中具有吸引力的封装载体[15]。酵母细胞微囊化已用于包封各种精油、酶、萜烯、生物碱、药物分子、纳米粒以至于活的微生物[16-18]。其中大部分封装载体是酿酒酵母。目前, 酿酒酵母微胶囊已被广泛应用于新型药物递送[19-21]。
由于酵母细胞具有良好的安全性, 其组分如细胞壁、细胞膜等也被广泛研究, 用于各种疾病的治疗[22]。例如, 酵母微胶囊被用作核酸药物与小分子药物的递送载体及口服疫苗的展示平台, 用于治疗癌症、炎症类疾病、糖尿病等[23-27]。Xu等[28]构建了一种酵母细胞壁来源的纳米制剂, 提高淋巴结引流效率, 逆转免疫抑制微环境, 从而有效抑制肿瘤生长。Zhou等[20]基于仿生酵母微胶囊包裹纳米粒, 靶向治疗急性和慢性炎症病毒感染及肿瘤。Zhang等[29]通过口服包裹IL-1β短发夹RNA酵母微胶囊用于创伤后骨关节炎的治疗。Lin等[30]利用酵母细胞膜包裹大肠杆菌Nissle 1917来增强其靶向性。酵母细胞膜可有效促进dectin-1受体介导的M细胞吞噬作用, 进而诱发强烈的黏膜免疫反应。
酵母细胞膜膜材料组成成分相对简单稳定, 制备过程简单。此外, 酵母细胞可实现大规模培养, 有望实现酵母细胞膜材料的批量制备。基于上述研究背景, 本研究以酵母细胞膜材料为研究对象, 构建酵母膜囊泡纳米系统, 将其作为药物载体, 分别包裹姜黄素(curcumin, Cur)和二氧化硅纳米粒, 并通过一系列体外实验来探究该载体系统的应用潜能。本研究将为酵母膜囊泡纳米系统的进一步生物医学应用提供新思路和方法。
材料与方法
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试剂  蛋白胨、酵母提取物、葡萄糖、蔗糖、氯化钠、乙二胺四乙酸二钠(EDTA-Na2)、Tris-HCl、CCK-8 (cell counting kit-8)试剂盒(细胞增殖及毒性检测试剂盒)、活/死细胞双染calcein-AM/PI试剂盒、BCA (bicinchoninic acid)蛋白浓度试剂盒、细胞膜红色荧光染色试剂盒(DiI)、活性氧检测试剂盒(DCFH-DA)、Hoechst 33258染色液、考马斯亮蓝染色套装(染色液+脱色液)(北京索莱宝科技有限公司); 2-巯基乙醇、咪唑、无水乙醇、十六烷基三甲基溴化铵、碳酸钠、甲醇、二甲基亚砜(DMSO)(上海麦克林生化科技股份有限公司); 蜗牛酶(上海源叶生物科技有限公司); 山梨醇(广州赛国生物科技有限责任公司)。RAW 264.7和Caco-2细胞来自本实验室储存。
仪器  多功能酶标仪(Multiskan FC, 美国Thermo Fisher Scientific公司); 透射电镜(Tecnai G2 Spirit Twin, FEI公司); 激光共聚焦显微镜(A1-ISTAR, 日本尼康公司); 紫外分光光度计(UV-3600, 日本岛津公司); 动态光散射仪(Mastersizer 3000, 美国马尔文公司)。
培养基的配制和菌株的培养  酵母膏胨葡萄糖(yeast extract peptone dextrose medium, YPD)液体培养基的配制: 称量10 g酵母提取物与20 g蛋白胨, 用ddH2O定容至900 mL, 121 ℃高压灭菌20 min。称取20 g葡萄糖溶于100 mL ddH2O, 105 ℃高压灭菌15 min。将葡萄糖溶液加入到上述900 mL培养基溶液中, 即获得YPD培养基。此外, 额外添加20 g琼脂粉, 即可配制YPD固体培养基。
实验菌株选用酿酒酵母(Saccharomyces cerevisiae)BY4741, 采用YPD培养基进行培养。首先从平板挑取单菌落, 加入YPD液体培养基, 30 ℃培养24 h。以1∶100比例接种至新培养基, 30 ℃继续培养24 h。
酵母细胞质膜的提取与表征  通过1 500 ×g离心收集过夜培养的1 L酿酒酵母。ddH2O洗涤3次, 加入50 mL预处理液(由10 mmol EDTA-Na2、10 mmol Tris-HCl、1 mol山梨醇及ddH2O配制1 L溶液, 随后加入100 μL 2-巯基乙醇, 得到预处理液)重悬, 于30 ℃条件下孵育1 h, 随后加入60 mg蜗牛酶孵育2 h去除细胞壁。在反应过程中, 每隔30 min取样并测定600 nm处吸光度变化, 以此判断酵母去除细胞壁的进程。通过光学显微镜观察原生质体的制备是否成功, 在光学显微镜下, 酵母菌呈白色, 细胞膜与细胞质分界处有明显界限, 而原生质体呈黑灰色, 且细胞膜分界模糊, 由此可分辨二者。为进一步判定细胞壁的去除效果, 用细胞膜红色荧光探针(DiI)和Hoechst 33342染色液对酵母菌和原生质体进行染色, 通过激光共聚焦显微镜来判断细胞壁是否完全去除。
将原生质体置于高压细胞破碎仪中, 1 500 bar压力下破碎7次得到细胞破碎液。6 500 ×g离心20 min去除细胞碎片, 35 000 ×g离心1 h获得粗膜提取物。100 000 ×g蔗糖密度梯度(1.1、1.65、2.25 mol·L-1)离心11 h, 用注射器获得1.65和2.25 mol·L-1蔗糖垫之间的细胞质膜, 1×磷酸盐缓冲液(phosphate buffered saline, PBS)重悬后-80 ℃保存。
将细胞破碎液、质膜粗提物及纯化的质膜进行聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色分析。采用BCA试剂盒测定蛋白浓度统一上样时蛋白浓度, 加入5× SDS-PAGE上样缓冲液, 99 ℃加热5 min使蛋白变性。设置电压80 V, 进行30 min电泳, 浓缩胶结束后调为电压120 V, 时间90 min。用考马斯亮蓝染色液室温染色1 h, 脱色液脱色8 h, 利用凝胶成像仪拍摄蛋白胶图像。
酵母质膜囊泡的制备与表征  酵母细胞质膜溶液经超声处理10 min。将细胞质膜溶液分别通过孔径为0.4 μm和0.2 μm的聚碳酸酯多孔膜挤压7次, 得到酵母质膜囊泡(Saccharomyces cerevisiae membrane vesicle, SMV)。利用动态光散射仪测定SMV的粒径分布和表面电位。
将SMV分散在PBS、胃模拟液(simulated gastric fluid, SGF)和肠模拟液(simulated intestinal fluid, SIF)中, 在4 ℃条件下储存, 于1、3、5、10天取样测量其粒径变化。
细胞毒性实验  将贴壁的RAW 264.7和Caco-2细胞分别接种于96孔板, 每孔接种约1×105个细胞, 每组平行6孔, 待细胞完全贴壁后更换含不同浓度SMV (RAW 264.7细胞: 12、25、50、100、150、200 μg·mL-1; Caco-2细胞: 10、20、40、80、160 μg·mL-1)的细胞培养基, 将培养板放在培养箱中预培养24 h。每孔加入10 μL CCK-8试剂, 培养箱中培养1 h, 用多功能酶标仪测量450 nm处吸光度。
向12孔板中贴壁的RAW 264.7细胞加入不同浓度SMV (12、50、100 μg·mL-1), 加入PBS作为空白对照。共孵育24 h后进行calcein-AM/PI活细胞/死细胞双染, 共聚焦显微镜观察检测活细胞(黄绿色荧光)及死细胞(红色荧光)。采用Image J软件对死细胞的荧光强度定量分析。
载药SMV的制备与表征  为了探究SMV的载药能力, 取2 mL蛋白浓度为500 μg·mL-1的SMV与16 mg Cur共孵育2 h, 35 000 ×g离心10 min, 得到载药SMV (SMV-Cur)。通过测定不同浓度Cur (2、4、8、12、16、20、40、80 μg·mL-1)在426 nm处的紫外吸收值来绘制标准曲线, 并计算SMV-Cur载药量。为了获得最大载药率, 本研究尝试了共孵育、反复冻融与超声3种方法。
药物包封率(%) = (W2 - W1)/W2 × 100%, 其中W1为游离药物含量, W2为投药总量。
体外药物释放: 将Cur浓度为1 mg·mL-1的SMV-Cur重悬于1 mL PBS (pH = 7.4)中, 在2、4、6、8、10、12 h不同时间点13 000 ×g离心并收集上清。利用紫外分光光度计测定每个时间点释放Cur的紫外吸收值, 并根据标准曲线来计算各时间点药物累积释放量。
SMV包覆二氧化硅(SMV@SiO2)的制备与表征  称取0.1 g的十六烷基三甲基溴化铵, 溶于25 mL水中, 随后加入6 mL无水乙醇和50 µL二乙醇胺。60 ℃搅拌反应30 min。缓慢注入2 mL正硅酸乙酯, 继续搅拌反应2 h。所得产物用无水乙醇清洗3次。将制备得到的二氧化硅纳米粒(SiO2)与SMV溶液混合, 用脂质体挤压器将其分别通过孔径为0.4 μm和0.2 μm的聚碳酸酯多孔膜挤压7次, 得到SMV@SiO2。通过透射电镜观察SiO2、SMV@SiO2的形貌。利用动态光散射仪来测定其粒径大小。
为了探究SMV是否成功包覆在SiO2表面, 利用BCA检测法来测定SiO2与SMV@SiO2溶液中蛋白含量。随后利用考马斯亮蓝染色来分析SMV、SiO2、SMV@SiO2的蛋白分布。
将SMV、SiO2、SMV@SiO2分散在PBS、胃模拟液和肠模拟液中, 将其置于4 ℃环境下, 于1、3、5、10、15天取样测量其粒径变化。
细胞摄取实验  首先, 将SMV与DiI染色液(1∶1 000)在室温条件下避光孵育30 min, 20 000 ×g离心3次除去多余染料。将贴壁的RAW 264.7细胞与50 μg·mL-1 SMV分别孵育0.5、1、5、12 h, 孵育结束后用Hoechst染色液进行细胞核染色, PBS清洗3次, 采用共聚焦显微镜分析细胞摄取效果。
活性氧检测实验  将RAW 264.7细胞悬液接种于24孔板中, 每孔接种约3×105个细胞, 过夜培养使细胞贴壁。分别加入20 μg Cur、负载相同浓度Cur的SMV-Cur及SMV, 30 min后加入脂多糖(LPS)诱导细胞炎症反应, 37 ℃孵育24 h。随后分别加入活性氧荧光探针DCFH-DA (1∶1 000)及Hoechst染色液(1∶100), 孵育30 min后用激光共聚焦显微镜观察细胞内绿色荧光的强弱。
统计学分析  数据均以平均值±标准误[x ± SEM (standard error of the mean)] 表示, 所有实验结果在相同条件下在至少3个独立实验中得到证实。使用GraphPad Prism 8进行统计分析, 统计学比较采用t检验, P < 0.05表示差异有统计学意义。
结果与讨论
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1 酵母细胞质膜的表征
向培养至对数期的酿酒酵母中加入预处理液和蜗牛酶, 酶解细胞壁, 以获得原生质体, 高压破碎原生质体后通过差速离心法及蔗糖密度梯度离心收集酵母质膜(图 1A)。预处理及酶解期间, 测定酵母悬浮液600 nm处的吸光度变化。结果表明: 吸光度随时间逐渐下降, 说明随着酶解反应的进行, 酵母细胞壁被逐渐消化(图 1B、C)。
为了更直观地观察原生质体, 首先用光学显微镜观察, 酵母细胞和原生质体之间有明显的形态差异(图 1D)。亲脂性染料DiI染色后, 共聚焦显微镜下可观察到原生质体细胞膜被DiI染色, 发出红色荧光(图 1E), 说明酶解法已成功制备酵母原生质体。
原生质体经高压破碎、差速离心以及蔗糖密度梯度离心后, 于1.65与2.25 mol·L-1蔗糖垫之间获得质膜产物(图 1F)。SDS-PAGE实验结果表明: 细胞质膜蛋白条带位置与预期相符。相较于细胞破碎液, 细胞质膜样品在150和30 kDa处出现细胞膜标志蛋白条带Drs2p及COX18, 并且蛋白含量明显提高(图 1G)。
2 SMV的表征
通过挤出法来制备酿酒酵母质膜囊泡SMV。透射电子显微镜(TEM)图像表明SMV成功制备, 大小在150~200 nm (图 2A)。粒径分布数据表明: SMV的水合粒径约为185.1 nm (图 2B)。此外, SMV表面带有负电荷, 表面电位约为-12.8 mV。为了探究SMV的稳定性, 将SMV分散在PBS溶液、肠模拟液、胃模拟液中。结果表明: 在5天内, SMV粒径大小没有明显变化(图 2C), 但随着时间延长, SMV在胃模拟液和肠模拟液中会发生聚集, 粒径增大。
3 SMV的细胞毒性
使用小鼠巨噬细胞RAW 264.7进行活/死细胞双染实验(图 3A、B)。活细胞被calcein-AM染色, 发出绿色荧光, 而死细胞被PI染色后呈红色荧光。结果表明: 随着SMV浓度增加, 红色荧光标记的死细胞逐渐增多, 说明SMV具有一定的细胞毒性。此外, 使用人克隆结肠腺癌细胞Caco-2 (图 3C)及RAW 264.7 (图 3D)进行CCK-8细胞毒性分析。结果表明: 与不同浓度的SMV共孵育24 h后, RAW 264.7细胞及Caco-2细胞活性均大于85%, 说明SMV具有良好的生物相容性。
4 SMV的细胞摄取
在细胞摄取实验中, SMV可被巨噬细胞有效摄取(图 4A)。当巨噬细胞与SMV共孵育0.5 h时, 细胞内开始出现红色荧光。随着共孵育时间延长, 红色荧光逐渐增强, 将SMV随时间逐渐被巨噬细胞摄取的过程可视化(图 4B)。
5 SMV@SiO2的表征
为了探究SMV包覆纳米粒的可能性, 选择将SMV包覆在SiO2表面。TEM图像表明(图 5A), SMV成功包覆在SiO2表面, 得到SMV@SiO2。动态光散射(DLS)结果表明: SMV的水和粒径为178.8 nm, SMV@SiO2的水和粒径大约为279.8 nm (图 5B)。通过BCA和考马斯亮蓝实验进一步验证, 酵母质膜成功包覆在纳米粒表面(图 5C、D)。随后探究了SMV@SiO2在不同溶液中的稳定性(图 5E), 在储存的10天内, SMV@SiO2的粒径均未发生明显变化, 说明其在模拟胃肠道的pH等条件下仍可保持较长时间的稳定性。
6 SMV包载Cur
Cur是从姜黄的根茎中提取的一种天然疏水生物活性物质, 具有多种生理活性(抗氧化、抗肿瘤、降糖、抗炎、抗菌、创面愈合等)。由于其抗炎和抗氧化作用, Cur在治疗炎症性肠病(inflammation bowel disease, IBD)上被视为有良好前景的候选药物。然而, Cur的水溶性和光热稳定较差, 在胃肠道有着吸收率低、易降解、代谢快等特性, 限制了其口服使用。因此本研究将SMV作为Cur药物递送的载体, 并初步探究了其抗炎效用。
Cur及包载Cur的酵母质膜囊泡SMV-Cur在423 nm处都有紫外吸收峰, 表明Cur已成功载入到SMV中(图 6A)。为了获得最高载药量, 本研究尝试通过共孵育法、超声法及反复冻融法将Cur装载到SMV中。载药量数据如图 6B所示, 共孵育法的药物包封率最高可达到17.2% ± 1.4%。药物释放实验结果表明(图 6C): 负载的Cur可缓慢从SMV中释放出来。
为了验证Cur、SMV与SMV-Cur的体外抗炎效果, 将巨噬细胞RAW 264.7与活性氧荧光探针DCFH-DA共孵育。结果表明: Cur与SMV-Cur可有效降低巨噬细胞产生的活性氧含量(图 6D), 有望用于肠炎的治疗。后续将进一步探究其在体内的应用效果。
结论
收起
综上, 本研究将酵母膜囊泡纳米系统作为药物载体, 在其内部负载化学药物姜黄素或二氧化硅纳米粒。酵母细胞膜可通过挤压形成形貌稳定、粒径均一的质膜囊泡SMV。SMV在肠胃模拟液中可稳定存在, 有望用于口服药物递送。此外, SMV细胞毒性较低, 具有良好的生物安全性。SMV可被巨噬细胞摄取, 通过包载姜黄素, 可有效降低炎症巨噬细胞内的活性氧含量, 有望用于炎症性肠病的口服治疗。同时, SMV可在二氧化硅颗粒表面形成纳米涂层, 有效提高纳米粒的稳定性, 在以后的研究中, 可用于构建仿生纳米粒。本研究有望为酵母膜囊泡纳米系统的构建提供新思路和方法。
作者贡献: 赵鹏程负责文章的初步构思、实验数据收集与处理及初稿撰写; 张英英负责实验指导、数据整理及论文修改; 张明慧负责部分数据的收集和处理; 王汉杰负责文章构思与论文修改。所有作者均参与手稿撰写与修改。
利益冲突: 所有作者声明无相关利益冲突。
基金
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  • 国家重点研发计划项目(2019YFA0906500)
  • 国家自然科学基金优秀青年科学基金项目(32122047)
  • 国家自然科学基金资助项目(31971300)
  • 国家自然科学基金资助项目(81771970)
  • 天津市重点研究与开发计划项目(19YFZCSY00190)
文献
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2023年第58卷第6期
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doi: 10.16438/j.0513-4870.2022-1264
  • 接收时间：2022-11-24
  • 首发时间：2025-11-21
  • 出版时间：2023-06-12
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  • 收稿日期：2022-11-24
  • 修回日期：2022-12-25
基金
国家重点研发计划项目(2019YFA0906500)
国家自然科学基金优秀青年科学基金项目(32122047)
国家自然科学基金资助项目(31971300)
国家自然科学基金资助项目(81771970)
天津市重点研究与开发计划项目(19YFZCSY00190)
作者信息
    1.天津大学生命科学学院, 天津 300072
    2.徐州医科大学医学影像学院, 江苏 徐州 221004

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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
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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