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Article(id=1198628608362250632, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198628599835227075, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-1347, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1670342400000, receivedDateStr=2022-12-07, revisedDate=1678204800000, revisedDateStr=2023-03-08, acceptedDate=null, acceptedDateStr=null, onlineDate=1763704929675, onlineDateStr=2025-11-21, pubDate=1686499200000, pubDateStr=2023-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763704929675, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763704929675, creator=13701087609, updateTime=1763704929675, 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=1677, endPage=1684, ext={EN=ArticleExt(id=1198628608790069689, articleId=1198628608362250632, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Preparation and preliminary evaluation of SARS-CoV-2 DNA vaccine based on PLGA nanoparticles, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

We constructed and optimized the plasmid DNA (pDNA) Opt-S encoding the gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, using poly (lactic-co-glycolic acid) copolymer (PLGA) as a delivery carrier for pDNA. PLGA-pDNA NPs were loaded by nanoprecipitation and its properties in vitro were preliminary evaluated. The results showed that the prepared PLGA-pDNA NPs were regular morphology, clear edges, with an average particle size of (184.2 ± 2.4) nm, polydisperse index (PDI) of 0.093 ± 0.013, zeta potential of (-68.10 ± 0.36) mV, and encapsulation rate of (98.92 ± 0.22)%. The PLGA-pDNA NPs were stable at -20 ℃ for 7 months and could protect pDNA against nuclease degradation. And they also exhibited sustained release of pDNA in vitro. The PLGA-pDNA NPs have low cytotoxicity and high safety. In addition, in vitro transfection experiments showed that the SARS-CoV-2 S gene could enter cells and be expressed. These results indicate that PLGA-pDNA NPs non-viral gene vector have simple preparation process and good performance, which are expected to provide a new idea for the research and development of SARS-CoV-2 vaccine.

, authors=null, authorsList=Ya-qi WU, Meng LI, Hao-nan XING, Da-quan CHEN, Ai-ping ZHENG, authorCompany=null, correspAuthors=Da-quan CHEN, Ai-ping ZHENG, 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=1198628611520561851, articleId=1198628608362250632, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于PLGA纳米粒的SARS-CoV-2 DNA疫苗的制备及初步评价, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本研究构建并优化编码新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)刺突(spike protein, S)蛋白基因序列的质粒DNA (plasmid DNA, pDNA)Opt-S, 以聚乳酸-羟基乙酸共聚物[poly (lactic-co-glycolic acid), PLGA] 纳米粒(nanoparticles, NPs)作为pDNA的递送载体, 采用纳米沉淀法制备PLGA-pDNA NPs, 对其体外性质进行初步评价。结果表明所制备的PLGA-pDNA NPs形态规整, 边缘清晰, 平均粒径为(184.2 ± 2.4) nm, 多分散系数(polydisperse index, PDI) 为0.093 ± 0.013, zeta电位为(-68.10 ± 0.36) mV, 包封率为(98.92 ± 0.22)%, 于-20 ℃储存7个月粒径和PDI变化幅度均较小, 稳定性较好, 能保护pDNA免受核酸酶降解, 并具有一定的缓释效果, 细胞毒性低, 安全性高, 体外转染实验表明SARS-CoV-2 S基因可以进入细胞并表达。以上结果表明PLGA-pDNA NPs非病毒基因载体制备工艺简单、性能良好, 有望为SARS-CoV-2疫苗研发提供新思路。

, authors=null, authorsList=吴雅琦, 李蒙, 邢昊楠, 陈大全, 郑爱萍, authorCompany=null, correspAuthors=陈大全, 郑爱萍, authorNote=null, correspAuthorsNote=
*陈大全, Tel: 86-535-6706021, E-mail: ;
郑爱萍, Tel: 86-10-66931694, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2023, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=EK+hrbCN5EbqfyYO5J4ujQ==, magXml=brE+BmKUEW4Vr95F8kq8mQ==, pdfUrl=null, pdf=z8CEf2ZHWok/mC7B/cBrGg==, pdfFileSize=2900636, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=+61O63xAFiZvtrUuLZmJIQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=Xh0hyNuOdMF6UhT+IgNxJQ==, mapNumber=null, fund=null)}, authors=[Author(id=1198960135151383472, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, 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=1198960135331738558, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, authorId=1198960135151383472, language=EN, stringName=Ya-qi WU, firstName=Ya-qi, middleName=null, lastName=WU, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=1, 2, address=1. Pharmacy School, Yantai University, Yantai 264005, China
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Int J Pharm, 2008, 354: 210-216., articleTitle=Establishing chitosan coated PLGA nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1198960134874559376, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, xref=null, ext=[AuthorCompanyExt(id=1198960134882947985, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, companyId=1198960134874559376, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. 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Lane 1: <i>Opt-S</i>; Lane M: Marker; B: Enzyme digestion results. Lane 1: <i>Nhe</i>I-<i>EcoR</i>I enzyme digestion; Lane M: Marker , figureFileSmall=jQ4BP5kZ1+hPyc0xwZWIbg==, figureFileBig=L+fF5Von5cmFiFUTxkMCLg==, tableContent=null), ArticleFig(id=1198960139454738739, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=Lc8DdTaInyiD/gy+xTw+nw==, figureFileBig=ujMFoPzyyaL/GbffXjDCrw==, tableContent=null), ArticleFig(id=1198960139563790653, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 2, caption= Visualization of the PLGA-pDNA NPs. Lane 1: PLGA/pDNA 36∶1; Lane 2: PLGA/pDNA 48∶1; Lane 3: PLGA/pDNA 72∶1; Lane 4: PLGA/pDNA 96∶1; Lane 5: <i>Opt-S</i>; Lane 6: PBS; Lane M: Marker , figureFileSmall=Lc8DdTaInyiD/gy+xTw+nw==, figureFileBig=ujMFoPzyyaL/GbffXjDCrw==, tableContent=null), ArticleFig(id=1198960139752534353, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=n3VAvxRWdX082Btz6Kt9mQ==, figureFileBig=8xjNgZiWa3H2nuJG362Vrw==, tableContent=null), ArticleFig(id=1198960139911917922, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 3, caption= Surface morphology and particle size distribution of PLGA NPs. 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Lane 1: PLGA-pDNA NPs with DNase Ⅰ; Lane 2: <i>Opt-S</i> pDNA with DNase Ⅰ; Lane 3: <i>Opt-S</i> pDNA without DNase Ⅰ; Lane M: Marker , figureFileSmall=8O3kH4lcPxoX3tHwSUwezQ==, figureFileBig=3yMjeeV+lJYvh/MODP+XRg==, tableContent=null), ArticleFig(id=1198960140415234462, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=WQs21AZt1XmYIpF9A62WbQ==, figureFileBig=j1YmtSXtur90h5xdIhlgRw==, tableContent=null), ArticleFig(id=1198960140570423728, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 5, caption= Stability of PLGA-pDNA NPs at 3 and 7 months (<i>n</i> = 3, <span class="mag-xml-inline-formula">$\bar{x}$</span> ± <i>s</i>) , figureFileSmall=WQs21AZt1XmYIpF9A62WbQ==, figureFileBig=j1YmtSXtur90h5xdIhlgRw==, tableContent=null), ArticleFig(id=1198960140717224382, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=EluIyJXWoTvsATzD/CHt7g==, figureFileBig=E0ZwTkmeuavt7Wu2lcF5ng==, tableContent=null), ArticleFig(id=1198960140901773776, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 6, caption= Cumulative release of pDNA from PLGA-pDNA NPs (<i>n</i> = 3, <span class="mag-xml-inline-formula">$\bar{x}$</span> ± <i>s</i>) , figureFileSmall=EluIyJXWoTvsATzD/CHt7g==, figureFileBig=E0ZwTkmeuavt7Wu2lcF5ng==, tableContent=null), ArticleFig(id=1198960141002437086, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=L/8jAvqHGR/QMjvXkN9W+Q==, figureFileBig=kpFnog2CtBJcwiKBi9aG0g==, tableContent=null), ArticleFig(id=1198960141115683311, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 7, caption= Blank PLGA NPs (A), PLGA-pDNA NPs and Lipo3000 (B) act on DC2.4 cell viability results. <i>n</i> = 6, <span class="mag-xml-inline-formula">$\bar{x}$</span> ± <i>s</i>. <sup>**</sup><i>P</i> < 0.01, <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=L/8jAvqHGR/QMjvXkN9W+Q==, figureFileBig=kpFnog2CtBJcwiKBi9aG0g==, tableContent=null), ArticleFig(id=1198960141224735231, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=oj8D8BH92aNttMEE4+R1Ug==, figureFileBig=pLMU815vT1o4PTFpytcXNw==, tableContent=null), ArticleFig(id=1198960141354758664, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Figure 8, caption= Evaluation of cell transfection <i>in vitro</i>. A: Results of immunofluorescence identification <i>in vitro</i>; B: Results of flow cytometry <i>in vitro</i> transfection; GFP: Green fluorescent protein , figureFileSmall=oj8D8BH92aNttMEE4+R1Ug==, figureFileBig=pLMU815vT1o4PTFpytcXNw==, tableContent=null), ArticleFig(id=1198960141484782104, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample name Size-average
/nm		 PDI Zeta potential
/mV
PLGA (50/50) NPs 101.3 ± 1.4 0.045 ± 0.010 -34.97 ± 2.65
PLGA (75/25) NPs 124.8 ± 1.2 0.049 ± 0.018 -7.47 ± 0.86
PLGA (85/15) NPs 119.7 ± 2.6 0.060 ± 0.020 -12.43 ± 0.59
), ArticleFig(id=1198960141627388454, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=CN, label=Table 1, caption=

PLGA model screening results. PLGA NPs: Poly (lactic-co-glycolic acid) nanoparticles; PDI: Polydisperse index

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample name Size-average
/nm		 PDI Zeta potential
/mV
PLGA (50/50) NPs 101.3 ± 1.4 0.045 ± 0.010 -34.97 ± 2.65
PLGA (75/25) NPs 124.8 ± 1.2 0.049 ± 0.018 -7.47 ± 0.86
PLGA (85/15) NPs 119.7 ± 2.6 0.060 ± 0.020 -12.43 ± 0.59
), ArticleFig(id=1198960141728051761, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198628608362250632, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample name Size-average
/nm		 PDI Zeta potential
/mV
PLGA 10 mg·mL-1 160.9 ± 1.5 0.138 ± 0.029 -41.60 ± 1.55
PLGA 20 mg·mL-1 155.3 ± 2.0 0.134 ± 0.005 -46.40 ± 0.26
PLGA 50 mg·mL-1 1 089.3 ± 687.7 0.916 ± 0.146 -24.20 ± 2.74
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PLGA concentration screening results

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Sample name Size-average
/nm		 PDI Zeta potential
/mV
PLGA 10 mg·mL-1 160.9 ± 1.5 0.138 ± 0.029 -41.60 ± 1.55
PLGA 20 mg·mL-1 155.3 ± 2.0 0.134 ± 0.005 -46.40 ± 0.26
PLGA 50 mg·mL-1 1 089.3 ± 687.7 0.916 ± 0.146 -24.20 ± 2.74
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PLGA/pDNA mass ratio Size-average
/nm		 PDI Zeta potential
/mV
36∶1 165.1 ± 2.3 0.146 ± 0.009 -5.70 ± 1.91
48∶1 197.5 ± 0.4 0.172 ± 0.034 -34.97 ± 8.54
72∶1 193.4 ± 3.4 0.121 ± 0.034 -44.20 ± 2.19
96∶1 259.5 ± 1.44 0.048 ± 0.028 -44.27 ± 7.71
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Screening results of PLGA/pDNA mass ratio. pDNA: Plasmid DNA

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PLGA/pDNA mass ratio Size-average
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48∶1 197.5 ± 0.4 0.172 ± 0.034 -34.97 ± 8.54
72∶1 193.4 ± 3.4 0.121 ± 0.034 -44.20 ± 2.19
96∶1 259.5 ± 1.44 0.048 ± 0.028 -44.27 ± 7.71
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基于PLGA纳米粒的SARS-CoV-2 DNA疫苗的制备及初步评价
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吴雅琦 1, 2 , 李蒙 2 , 邢昊楠 2 , 陈大全 1, * , 郑爱萍 2, *
药学学报 | 研究论文 2023,58(6): 1677-1684
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药学学报 |研究论文 2023 , 58 (6) : 1677 -1684
基于PLGA纳米粒的SARS-CoV-2 DNA疫苗的制备及初步评价
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吴雅琦1, 2, 李蒙2, 邢昊楠2, 陈大全1, * , 郑爱萍2, *
作者信息
  • 1.烟台大学药学院, 山东 烟台 264005
  • 2.军事科学院军事医学研究院毒物药物研究所, 北京 100850
通讯作者:
*陈大全, Tel: 86-535-6706021, E-mail: ;
郑爱萍, Tel: 86-10-66931694, E-mail:
Preparation and preliminary evaluation of SARS-CoV-2 DNA vaccine based on PLGA nanoparticles
Ya-qi WU1, 2, Meng LI2, Hao-nan XING2, Da-quan CHEN1, * , Ai-ping ZHENG2, *
Affiliations
  • 1. Pharmacy School, Yantai University, Yantai 264005, China
  • 2. Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China
出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1347
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摘要
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本研究构建并优化编码新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)刺突(spike protein, S)蛋白基因序列的质粒DNA (plasmid DNA, pDNA)Opt-S, 以聚乳酸-羟基乙酸共聚物[poly (lactic-co-glycolic acid), PLGA] 纳米粒(nanoparticles, NPs)作为pDNA的递送载体, 采用纳米沉淀法制备PLGA-pDNA NPs, 对其体外性质进行初步评价。结果表明所制备的PLGA-pDNA NPs形态规整, 边缘清晰, 平均粒径为(184.2 ± 2.4) nm, 多分散系数(polydisperse index, PDI) 为0.093 ± 0.013, zeta电位为(-68.10 ± 0.36) mV, 包封率为(98.92 ± 0.22)%, 于-20 ℃储存7个月粒径和PDI变化幅度均较小, 稳定性较好, 能保护pDNA免受核酸酶降解, 并具有一定的缓释效果, 细胞毒性低, 安全性高, 体外转染实验表明SARS-CoV-2 S基因可以进入细胞并表达。以上结果表明PLGA-pDNA NPs非病毒基因载体制备工艺简单、性能良好, 有望为SARS-CoV-2疫苗研发提供新思路。

新型冠状病毒肺炎  /  新型冠状病毒DNA疫苗  /  聚乳酸-羟基乙酸共聚物  /  纳米沉淀法  /  非病毒基因载体

We constructed and optimized the plasmid DNA (pDNA) Opt-S encoding the gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, using poly (lactic-co-glycolic acid) copolymer (PLGA) as a delivery carrier for pDNA. PLGA-pDNA NPs were loaded by nanoprecipitation and its properties in vitro were preliminary evaluated. The results showed that the prepared PLGA-pDNA NPs were regular morphology, clear edges, with an average particle size of (184.2 ± 2.4) nm, polydisperse index (PDI) of 0.093 ± 0.013, zeta potential of (-68.10 ± 0.36) mV, and encapsulation rate of (98.92 ± 0.22)%. The PLGA-pDNA NPs were stable at -20 ℃ for 7 months and could protect pDNA against nuclease degradation. And they also exhibited sustained release of pDNA in vitro. The PLGA-pDNA NPs have low cytotoxicity and high safety. In addition, in vitro transfection experiments showed that the SARS-CoV-2 S gene could enter cells and be expressed. These results indicate that PLGA-pDNA NPs non-viral gene vector have simple preparation process and good performance, which are expected to provide a new idea for the research and development of SARS-CoV-2 vaccine.

corona virus disease 2019  /  SARS-CoV-2 DNA vaccine  /  poly (lactic-co-glycolic acid) copolymer  /  nanoprecipitation method  /  non-viral gene carrier
吴雅琦, 李蒙, 邢昊楠, 陈大全, 郑爱萍. 基于PLGA纳米粒的SARS-CoV-2 DNA疫苗的制备及初步评价. 药学学报, 2023 , 58 (6) : 1677 -1684 . DOI: 10.16438/j.0513-4870.2022-1347
Ya-qi WU, Meng LI, Hao-nan XING, Da-quan CHEN, Ai-ping ZHENG. Preparation and preliminary evaluation of SARS-CoV-2 DNA vaccine based on PLGA nanoparticles[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1677 -1684 . DOI: 10.16438/j.0513-4870.2022-1347
正文
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2019年底由新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)引起的新型冠状病毒肺炎(corona virus disease 2019, COVID-19)疫情迅速蔓延全球, 接种疫苗是控制疫情和预防死亡的最佳策略[1-5]。SARS-CoV-2主要由四种结构蛋白组成, 刺突(spike protein, S)蛋白负责介导病毒进入宿主细胞, 在此过程中跨膜丝氨酸蛋白酶2 (transmembrane protease serine 2, TMPRSS2)活化S蛋白, 促使S蛋白与细胞表面的血管紧张素转化酶2 (angiotensin-converting enzyme 2, ACE2)受体结合而进入人体, 因此S蛋白是病毒中和抗体的关键靶点[6-10], 针对S蛋白的多个疫苗平台被开发, 包括灭活疫苗、病毒减毒疫苗、病毒载体疫苗、亚单位疫苗和核酸疫苗等, 其中DNA疫苗研发速度快、生产制备简单、稳定性高, 同时可以诱导体液免疫和细胞免疫, 持续表达靶抗原, 不良反应少, 安全性好, 有望在新冠领域发挥重要作用[11-13]。
为提高S蛋白的表达水平, 对编码SARS-CoV-2 S蛋白的基因序列进行优化, 如: 提高GC含量、使用S-2P技术以及将S蛋白原始信号肽更改为组织纤溶酶原激活物(tissue plasminogen activator, tPA)信号肽等, 使用优化后的基因序列构建重组质粒Opt-S。裸DNA易被降解, 使DNA疫苗表达水平有限, 导致免疫原性弱[14, 15], 故选择具有良好生物相容性和生物可降解性的聚乳酸-羟基乙酸共聚物[poly (lactic-co-glycolic acid), PLGA] 作为质粒DNA (plasmid DNA, pDNA)载体, PLGA安全性高, 已被FDA批准使用, 能靶向黏膜表面, 提高M细胞的吞噬能力, 可以快速实现溶酶体逃逸, 并具有药物缓释性, 控制抗原缓慢释放, 持续产生免疫应答[16-18]。本研究采用纳米沉淀法制备PLGA-pDNA NPs, 对纳米粒进行处方筛选与表征, 同时对PLGA-pDNA NPs的抗脱氧核糖核酸酶Ⅰ(deoxyribonuclease Ⅰ, DNase Ⅰ)能力、稳定性、体外释放、体外表达水平及安全性进行初步评价, 为SARS-CoV-2 DNA疫苗的研发奠定理论与实践基础。
材料与方法
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试剂  PLGA (端羧基, 50/50, LP1299; 75/25, 0002084262, 德国Evonik公司); PLGA (端羧基, 85/15, P0097310, 广州帝奇医药技术有限公司); 泊洛沙姆407 (Pluronic F127, GND32821B, 武汉新大地环保材料股份有限公司); 二甲基亚砜(DMSO, RH155103, 上海易恩化学技术有限公司); LB液体培养基(PM302126160)、氨苄青霉素(SL32111420) (北京酷来搏科技有限公司); 无内毒素质粒大提试剂盒(W0110, 天根生化科技有限公司); 4%多聚甲醛(210315S)、Dil (D8700)、Hochest 33258 (B8030) (北京索莱宝科技有限公司); DNase Ⅰ (D7073)、乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA, ST066)、CCK-8试剂盒(C0038) (上海碧云天生物技术有限公司); PicoGreenTM dsDNA定量检测试剂(2383786)、Opti-MEM培养基(31985070)、胎牛血清(fetal bovine serum, FBS, 10100147C) (美国Thermo公司); 青霉素-链霉素(WH0621K61, 武汉普诺赛生命科技有限公司); DMEM培养基(319-070-CL, 维森特生物技术有限公司); SARS-CoV-2 spike antibody, rabbit PAb (40591-T62, 北京义翘神州生物技术有限公司); Alexa Fluor®647偶联试剂盒—Lightning-Link® (ab269823, 英国Abcam公司); DC2.4细胞(中国典型培养物保藏中心); 重组质粒由北京擎科生物有限公司合成。
仪器  立式压力蒸汽灭菌器(LS-B50L, 江阴滨江医疗设备有限公司); 恒温摇床(HNY-200D, 天津欧诺仪器股份有限公司); 恒温磁力搅拌器(SHJ-4B, 常州高德仪器制造有限公司); 冷冻离心机(5804R, 德国Eppendorf公司); NanoDrop Lite分光光度计(MD-L27E)、多功能酶标仪(Varioskan Lux)、细胞培养箱(Forma series Ⅱ) (美国Thermo公司); 马尔文粒径仪(Nano-ZS90, 英国Malvern公司); 透射电镜(H-7650, 日本HITACHI公司); 凝胶成像仪(5200Multi, 上海天能生命科学有限公司); 光学显微镜(CKX41, 日本OLYMPUS公司); 激光共聚焦显微镜(LSM880, 德国Carl Zeiss公司); 流式细胞仪(BD FACS ArianaTM Ⅱ, 美国BD公司)。
重组质粒的构建与鉴定
基因序列的设计  SARS-CoV-2 Opt-S重组质粒设计毒株选择武汉株, 将SARS-CoV-2 S基因序列进行优化, 低频密码子更换为高频密码子, 提高GC含量增强稳定性, 采用S-2P技术添加两个脯氨酸突变稳定S蛋白构象, 将S蛋白原始信号肽更改为tPA信号肽, 提高S蛋白的表达水平, 同时在翻译起始密码子前加入Kozak序列增强S基因翻译效率。
质粒的合成与鉴定  将优化的S基因序列片段插入pcDNA3.1载体, 构建重组质粒Opt-S, 酶切位点为NheI和EcoRI, 同时在Opt-S基因序列后添加一段绿色荧光蛋白(green fluorescent protein, GFP)基因序列, 构建Opt-S-GFP质粒, 便于后续实验观察, 设计完成后交由北京擎科生物有限公司合成。将合成后的质粒穿刺菌进行复苏得到含Opt-S质粒的大肠杆菌, 接种于经高压灭菌后加入氨苄青霉素的LB液体培养基中, 于37 ℃、180 r·min-1培养12~16 h, 使用无内毒素质粒大提试剂盒提取质粒。质粒提取后进行酶切鉴定、琼脂糖凝胶电泳评价及全质粒测序, 同时使用NanoDrop Lite分光光度计测定质粒浓度及纯度。
纳米粒的制备
空白PLGA纳米粒的制备  采用改进的纳米沉淀法[19]制备空白PLGA纳米粒。在磁力搅拌下, 将溶解于DMSO的PLGA溶液缓慢滴加至0.5% (w/v)的Pluronic F127水溶液中, 室温搅拌5 h (800 r·min-1), 然后将整个分散体系于4 ℃、15 000 r·min-1离心30 min, 使用磷酸盐缓冲溶液(phosphate-buffered saline, PBS, pH 7.4)重悬离心后沉淀, 过0.22 μm滤膜收集纳米粒。
PLGA-pDNA NPs的制备  除在溶解于DMSO的PLGA溶液中加入pDNA溶液外, 其余步骤与空白PLGA纳米粒的制备相同。
纳米粒的处方筛选  对PLGA型号、浓度及PLGA/pDNA质量比进行筛选, 考察不同型号PLGA (50/50、75/25、85/15), 不同浓度PLGA (10、20、50 mg·mL-1)以及PLGA/pDNA不同质量比(36∶1、48∶1、72∶1和96∶1)对所制备纳米粒理化性质的影响。分别取制备后适量纳米粒分散于去离子水中, 通过动态光散射法(dynamic light scattering, DLS)使用马尔文粒径仪测定纳米粒的粒径、多分散系数(polydisperse index, PDI)及zeta电位, 同时, 采用凝胶阻滞实验[20]进一步筛选PLGA/pDNA质量比(36∶1、48∶1、72∶1和96∶1), 进行0.6%琼脂糖凝胶电泳, 在凝胶成像仪下观察结果。
纳米粒的表征
形态观察  取少量纳米粒溶液滴至专用铜网上, 风干后滴加2% (w/v)磷钨酸负染2 min, 用滤纸吸去染色液, 待干后于透射电子显微镜(transmission electron microscope, TEM)下观察纳米粒形态。
粒径分布及zeta电位测定  取适量纳米粒分散于去离子水中, 使用马尔文粒径仪测定纳米粒的粒径及PDI, 并对其表面zeta电位进行检测。
包封率检测  采用PicoGreenTM dsDNA定量检测试剂[21]进行包封率评价, 按照PicoGreen染液: 1×TE缓冲液为1∶200的比例配制PicoGreen工作液。标准品工作液浓度设置为: 2 000、1 500、1 000、500、200、100和0 ng·mL-1, 使用1×TE缓冲液对标准品工作液及PLGA-pDNA NPs离心后上清液进行稀释, 分别加入相同体积的PicoGreen工作液, 室温避光孵育5 min, 从中取200 μL溶液转移至微量检测皿(96孔板)中, 平行配置三组(n = 3)。使用多功能酶标仪检测激发波长480 nm、发射波长520 nm处的荧光值, 绘制浓度与对应荧光强度的标准曲线, 以此计算上清液中游离DNA含量, 计算包封率(%) = (总pDNA含量-游离pDNA含量)/总pDNA含量×100%。
抗DNase Ⅰ能力考察  为验证PLGA-pDNA NPs对pDNA的保护作用, 取相应体积PLGA-pDNA NPs溶液及裸Opt-S质粒溶液, 分别加入5 μL DNase Ⅰ (1 U·μL-1), 37 ℃水浴孵育30 min, 向上述反应体系中加入2 μL EDTA, 65 ℃孵育10 min以失活DNase Ⅰ, 终止反应。取反应后复合物进行0.6%琼脂糖凝胶电泳, 同时设置不添加DNase Ⅰ的裸Opt-S质粒组, 电泳结束后在凝胶成像仪下观察质粒降解情况。
稳定性考察  将制备的PLGA-pDNA NPs于-20 ℃放置3或7个月后, 观察纳米粒的变化情况, 肉眼观察纳米粒是否发生聚集沉降, 通过马尔文粒径仪检测PLGA-pDNA NPs的粒径、PDI及zeta电位。
体外释放评价  采用恒温振荡摇床法对PLGA-pDNA NPs进行体外释放评价[22]。将制备的PLGA-pDNA NPs使用pH7.4的PBS重悬, 均分为三份于EP管中(n = 3), 于37 ℃恒温水浴中120 r·min-1振荡释放。在预设时间点(1、9、12、24 h和2、3、4、5、7、9、11、13、15、18和20天)取样, 同时补充与所取样品相同体积的PBS至原EP管中, 将所取样品于4 ℃、15 000 r·min-1离心20 min, 使用PicoGreenTM dsDNA定量检测试剂测定上清液的荧光强度, 计算pDNA释放量, 并绘制释放曲线。
体外细胞评价
细胞毒性实验  将DC2.4细胞以细胞数1×104/孔接种于96孔板, 培养至贴壁后, 分别加入不同浓度的空白PLGA NPs和PLGA-pDNA NPs, 空白PLGA NPs组浓度设置为2、5、10、20、40和80 mg·mL-1, PLGA-pDNA NPs及Lipo3000组pDNA浓度设置为0.1、0.5、1、2、4和8 μg·mL-1, 同时设置对照组和空白组, 对照组加入细胞和培养基, 空白组只加入培养基, 每组设置6个复孔, 分别孵育24和48 h后, 每孔加入10 μL CCK-8, 继续孵育2 h, 在酶标仪下检测波长450 nm处各孔的吸光度值(OD值), 计算细胞存活率= (实验组OD值-空白组OD组)/(对照组OD值-空白组OD组)×100%。
体外转染实验  将DC2.4细胞以细胞数1×106/孔接种于共聚焦小皿/6孔板, 37 ℃培养至细胞贴壁后进行转染实验, 将细胞分为PLGA-pDNA NPs组、Lipo3000组、裸Opt-S-GFP/裸Opt-S质粒组及空白对照组, 每组分别加入Opti-MEM培养基共孵育8 h后, 吸弃旧液, 加入DMEM完全培养基(10% FBS、1%青霉素-链霉素)继续培养。接种于共聚焦小皿的细胞转染48 h后, 使用4%多聚甲醛溶液固定细胞, Hochest 33258染色细胞核, Dil染色细胞膜, 进行激光共聚焦观察。接种于6孔板的细胞转染48 h后, 使用Alexa Fluor®647标记的SARS-CoV-2 S抗体进行胞内破膜染色1 h, 通过流式细胞术检测分析, 对纳米粒的转染情况进行评价。
统计学分析  采用SPSS 25软件进行数据分析, 数据采用$\bar{x}$ ± s表示, 两组间比较采用t检验, P < 0.05表示差异具有统计学意义。
结果
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1 重组质粒的构建与鉴定
采用琼脂糖凝胶电泳对质粒进行评价, 如图 1A所示, 所提取质粒显示出目标条带, 如图 1B所示, 经NheI-EcoRI首尾酶切后限制性酶谱显示两个大小分别为3 894 bp和5 371 bp的条带, 表明插入片段大小与预计结果相符, 酶切结果正确, 使用NanoDrop Lite分光光度计测定质粒浓度及纯度, 浓度≥ 500 ng·μL-1, A260/A280在1.8~2.0内, 符合要求, 同时全质粒测序成功, 表明重组质粒Opt-S成功构建。
2 纳米粒的制备与处方筛选
2.1 PLGA型号筛选
对不同PLGA型号的PLGA NPs筛选结果见表 1, 采用PLGA 50/50制备的纳米粒, 粒径和PDI均为三者中最小, 分散更均匀, zeta电位绝对值更高, 更稳定, 综合考虑, 选择PLGA 50/50型号。
2.2 PLGA浓度筛选
对不同PLGA浓度的PLGA NPs筛选结果见表 2, PLGA浓度在20 mg·mL-1时, 粒径相较更小, PDI < 0.2, 分散性良好, zeta电位更稳定, 因此, 选择PLGA浓度为20 mg·mL-1。
2.3 PLGA/pDNA质量比筛选
不同质量比PLGA-pDNA NPs的粒径、PDI及电位结果见表 3, 可以看出粒径和zeta电位绝对值有随PLGA/pDNA质量比增大而增加的趋势, PDI < 0.2, 粒径分布均匀, 综合来看, 质量比为72∶1时粒径更小, zeta电位相对较高, 采用凝胶阻滞实验进行进一步验证。
2.4 凝胶阻滞实验
不同质量比PLGA-pDNA NPs的凝胶阻滞结果如图 2所示, 与裸Opt-S质粒组相比, 经PLGA包裹的纳米粒组未在泳道上显示条带, 表明PLGA-pDNA NPs成功包载Opt-S。其中PLGA/pDNA质量比为72∶1和96∶1时制备的PLGA-pDNA NPs可以明显看出能将质粒滞留在加样孔, 且72∶1时亮度显著, 说明此质量比下PLGA-pDNA NPs能与DNA有效结合。综上所述, 确定PLGA/pDNA质量比为72∶1。
3 纳米粒的表征
3.1 TEM及DLS表征
空白PLGA NPs与PLGA-pDNA NPs的TEM及DLS表征见图 3, 通过TEM观察可见纳米粒形态规则, 表面光滑, 大小均匀, 边缘清晰。通过DLS检测可见粒径分布均匀, 空白PLGA NPs平均粒径为(107.6 ± 0.15) nm, PDI为0.042 ± 0.012, zeta电位为(-35.9 ± 1.25) mV, 处方优化工艺制备的PLGA-pDNA NPs平均粒径为(184.2 ± 2.4) nm, PDI为0.093 ± 0.013, zeta电位为(-68.10 ± 0.36) mV。
3.2 包封率检测
采用PicoGreenTM dsDNA定量检测试剂进行pDNA浓度-荧光强度标准曲线的绘制和包封率的测定, 由标准曲线得到DNA线性回归方程y = 0.030 2x-0.315 1 (r = 0.999 6), r > 0.999符合要求, 根据该回归方程计算PLGA-pDNA NPs中游离DNA的含量, 进而得出包封率为(98.92 ± 0.22)%。
4 抗DNase Ⅰ能力考察
考察PLGA-pDNA NPs对pDNA的核酸酶保护作用, 如图 4所示, 不添加DNase Ⅰ的裸Opt-S质粒组出现条带, PLGA-pDNA NPs包裹的Opt-S质粒经DNase Ⅰ作用后滞留在加样孔, 可看到加样孔中有明亮条带, 而经DNase Ⅰ作用后未被包裹的裸Opt-S质粒组加样孔及泳道中均无条带, 说明裸Opt-S质粒被DNase Ⅰ降解, PLGA-pDNA NPs可以保护Opt-S质粒不被DNase Ⅰ降解。
5 稳定性考察
PLGA-pDNA NPs于-20 ℃放置3或7个月后, 肉眼观察未见溶液发生聚集和沉降现象, 粒径和PDI变化如图 5所示, 3个月PLGA-pDNA NPs平均粒径为(207.1 ± 1.5) nm, PDI为0.117 ± 0.010, zeta电位为(-57.67 ± 2.55) mV; 7个月PLGA-pDNA NPs平均粒径为(228.5 ± 3.3) nm, PDI为0.132 ± 0.056, zeta电位为(-44.20 ± 4.42) mV, 可见纳米粒的粒径和PDI变化幅度均较小, 稳定性较好。
6 体外释放评价
PLGA-pDNA NPs的释放结果如图 6所示, PLGA-pDNA NPs中pDNA在0~48 h快速释放, 释放量达到(49.42 ± 0.50)%, 此为突释阶段, 随着时间的延长释放缓慢增加, 到20天释放达(64.83 ± 0.85)%, 具有一定的缓释效能。
7 体外细胞评价
7.1 细胞毒性实验
采用CCK-8法测定不同浓度的空白PLGA NPs及PLGA-pDNA NPs对DC2.4细胞作用24、48 h的细胞毒性, 并以Lipo3000作用24 h的细胞毒性作为对照。如图 7所示, 空白PLGA NPs和PLGA-pDNA NPs在不同浓度下的细胞存活率均大于80%, pDNA浓度≥ 1 μg·mL-1时, PLGA-pDNA NPs细胞毒性显著低于Lipo3000, 表明优化工艺制备的PLGA-pDNA NPs对DC2.4细胞无抑制作用, 无明显毒性, 构建的SARS-CoV-2 DNA疫苗纳米粒递送系统具有较高的安全性。
7.2 体外转染实验
将DC2.4细胞转染48 h后分别进行激光共聚焦观察和流式细胞术分析, 由图 8A可以看出, 空白PLGA NPs未能检测到绿色荧光, 裸Opt-S-GFP质粒组荧光较弱, PLGA-pDNA NPs组可以检测到较强于裸质粒组、偏弱于Lipo3000组的绿色荧光。由图 8B可以看出, 与空白组荧光强度相比, 裸Opt-S质粒组变化不大, 而PLGA-pDNA NPs组与Lipo3000组荧光强度均明显增加, PLGA-pDNA NPs组阳性率为55.8%, Lipo3000组阳性率为64.7%, 结果表明PLGA-pDNA NPs可以使SARS-CoV-2 Opt-S基因进入细胞并表达。
讨论
收起
COVID-19疫情给全球带来不同程度的影响, 对经济增长、社会公共健康造成了巨大冲击, 进而引发社会危机和心理危机, 随着各种更强传播性变异株的出现, 继续开发新的疫苗平台、使用不同的疫苗开发策略进一步提高疫苗的稳定性、有效性和安全性迫在眉睫。
本文基于PLGA纳米粒构建SARS-CoV-2 DNA疫苗, 选择SARS-CoV-2 S蛋白为疫苗靶点, 设计合成SARS-CoV-2重组质粒Opt-S, 使用具有生物相容性及生物可降解性等诸多优势的PLGA制备PLGA-pDNA NPs, 改善DNA易降解问题, 通过处方筛选, 控制PLGA-pDNA NPs的粒径、PDI及zeta电位于适宜的范围内, 确定了PLGA-pDNA NPs的最佳制备工艺。使用优化工艺制备的PLGA-pDNA NPs形态规整, 平均粒径为(184.2 ± 2.4) nm, 分散性较好, 具有较高的包封率和稳定性, 能抵抗DNase Ⅰ降解, 有一定的缓释效果, 体外细胞转染效率高于裸Opt-S质粒, 略低于Lipo3000, 但其转染效果不受血清和抗生素的影响, 细胞毒性低于Lipo3000, 安全性高, 表明PLGA-pDNA NPs可以更安全稳定地递送SARS-CoV-2 S基因进入细胞并表达, 为COVID-19疫苗的开发提供参考, 有望在应对COVID-19等传染病中发挥作用。
作者贡献: 吴雅琦是本研究的实验设计者和实验研究的执行人, 完成数据分析、论文初稿的撰写; 李蒙、邢昊楠指导并参与实验设计和结果分析; 陈大全、郑爱萍是项目构思者及负责人, 指导实验设计、数据分析、论文写作与修改。全体作者都阅读并同意最终的文本。
利益冲突: 所有作者均声明不存在任何利益冲突。

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doi: 10.16438/j.0513-4870.2022-1347
  • 接收时间：2022-12-07
  • 首发时间：2025-11-21
  • 出版时间：2023-06-12
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  • 收稿日期：2022-12-07
  • 修回日期：2023-03-08
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    1.烟台大学药学院, 山东 烟台 264005
    2.军事科学院军事医学研究院毒物药物研究所, 北京 100850

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2种不同金属材料的力学参数
科
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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