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Article(id=1218291751266210062, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, articleNumber=1001-2494(2024)13-1219-07, orderNo=null, doi=10.11669/cpj.2024.13.006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1673452800000, receivedDateStr=2023-01-12, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768392988268, onlineDateStr=2026-01-14, pubDate=1720368000000, pubDateStr=2024-07-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768392988268, onlineIssueDateStr=2026-01-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768392988268, creator=13701087609, updateTime=1768392988268, updator=13701087609, issue=Issue{id=1218291750003724554, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='13', pageStart='1173', pageEnd='1272', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768392987967, creator=13701087609, updateTime=1768394537396, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1218298248834503031, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1218298248838697336, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1219, endPage=1225, ext={EN=ArticleExt(id=1218291751484313871, articleId=1218291751266210062, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Preparation and Antibacterial Activity of Antimicrobial Peptide PMAP-24KK Hydrogel, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To prepare hydrogels containing antimicrobial peptide PMAP-24KK and evaluate antibacterial activity. METHODS The antimicrobial peptide PMAP-24KK was combined with sodium alginate, calcium carbonate, and gluconic acid-δ-lactone to form the PMAP-24KK hydrogel. Firstly, the formation time, swelling rate, moisture content, water retention rate, and release rate of PMAP-24KK hydrogel were analyzed. Then, the effect of PMAP-24KK hydrogel on the antibacterial activity in vitro, the sustained release, and the antibacterial barrier were determined by drug sensitivity test and skin wound healing in mice was analyzed. RESULTS The results showed that the PMAP-24KK hydrogel could form a gel state with network structure in about 2 min, and the swelling rate and moisture content reached equilibrium at 10 h, which were (158.83±3.26)% and (61.01±2.41)%, respectively. The water retention rate and release rate were about 55% and 81% after 24 h, respectively. The PMAP-24KK hydrogel showed the inhibitory effect on Staphylococcus aureus, Listeria monocytogenes, Salmonella choleraesuis, and Salmonella typhimurium. The sustained-release activity of PMAP-24KK hydrogel had no significant difference in 18 h, and gradually decreased after 18 h to 36 h without antibacterial activity. The PMAP-24KK hydrogel can effectively act as an antibacterial barrier and contribute to wound healing in mouse wound model. CONCLUSION Antimicrobial peptide PMAP-24KK hydrogel with good physicochemical properties and antibacterial activity is successfully prepared in this study, and it has good sustained-release and antibacterial barrier and promote wound healing, the study laid a foundation for further application research of antimicrobial peptide hydrogel.

, correspAuthors=Chengshui LIAO, 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=Fukang MAO, Wenqi FAN, Siyang LU, Junhui WANG, Endian WANG, Jingjing CUI, Xiaoli WANG, Mingfu NIU, Chengshui LIAO), CN=ArticleExt(id=1218291754038645049, articleId=1218291751266210062, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=抗菌肽PMAP-24KK水凝胶的制备及抗菌活性研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 制备含猪源抗菌肽-24KK(porcine myeloid antimicrobial peptide-24KK,PMAP-24KK)的水凝胶,评价其理化性能和抗菌活性。方法 将PMAP-24KK与海藻酸钠、碳酸钙和葡萄糖酸内酯结合得到PMAP-24KK水凝胶,首先分析PMAP-24KK水凝胶的形成时间、溶胀率、含水率、保水率和释放率等理化性质,然后进行药敏试验测定其对细菌的体外抗菌活性、缓释和抗菌屏障以及对小鼠皮肤创伤愈合的效果。结果 PMAP-24KK水凝胶在2 min左右可以形成具有网状结构的凝胶态,溶胀率和含水率均在10 h达到平衡状态,分别为(158.83±3.26)%和(61.01±2.41)%,24 h的保水率和释放率约为55%和81%。PMAP-24KK水凝胶对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌表现出抑菌效果。PMAP-24KK水凝胶18 h内的缓释作用没有明显区别,18 h后开始逐渐下降至36 h无抗菌活性。PMAP-24KK水凝胶能有效地起到抗菌屏障作用,且有助于小鼠创伤模型皮肤伤口的愈合。结论 本研究成功制备获得具有良好理化特性和抗菌活性的PMAP-24KK水凝胶,且具有较好的缓释和抗菌屏障以及促进伤口愈合的作用,为抗菌肽水凝胶应用的进一步研究奠定基础。

, correspAuthors=廖成水, authorNote=null, correspAuthorsNote=
* 廖成水,男,副教授,博士 研究方向:食源性微生物感染与新型药物开发研究 Tel:(0379)64563979
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毛付康,男,硕士研究生 研究方向:食源性微生物感染与新型药物开发研究

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毛付康,男,硕士研究生 研究方向:食源性微生物感染与新型药物开发研究

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毛付康,男,硕士研究生 研究方向:食源性微生物感染与新型药物开发研究

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Macromol Biosci, 2020, 20(2): e1900303., articleTitle=A spray-filming self-healing hydrogel fabricated from modified sodium alginate and gelatin as a bacterial barrier, refAbstract=null)], funds=[Fund(id=1218291760653062778, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, awardId=182102110061, language=CN, fundingSource=河南省科技攻关项目(182102110061), fundOrder=null, country=null), Fund(id=1218291760728560253, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, awardId=13450009, language=CN, fundingSource=河南科技大学青年骨干教师培养计划(13450009), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1218291754244165950, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, xref=1, ext=[AuthorCompanyExt(id=1218291754252554559, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754244165950, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 Laboratory of Functional Microorganisms and Animal Health, Henan University of Science and Technology, Luoyang 471023, China), AuthorCompanyExt(id=1218291754256748864, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754244165950, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 河南科技大学功能微生物与畜禽健康实验室, 河南 洛阳 471023)]), AuthorCompany(id=1218291754323857730, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, xref=2, ext=[AuthorCompanyExt(id=1218291754332246339, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754323857730, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 Key Laboratory of Live Carrier Biomaterials and Animal Disease Prevention and Control in Luoyang, Luoyang 471023, China), AuthorCompanyExt(id=1218291754340634948, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754323857730, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 洛阳市活载体生物材料与动物疫病防控重点实验室, 河南 洛阳 471023)]), AuthorCompany(id=1218291754411938121, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, xref=3, ext=[AuthorCompanyExt(id=1218291754420326730, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754411938121, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 College of Basic Medicine and Forensic Medicine, Henan University of Science and Technology, Luoyang 471023, China), AuthorCompanyExt(id=1218291754424521035, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754411938121, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=3 河南科技大学基础医学与法医学院, 河南 洛阳 471023)]), AuthorCompany(id=1218291754500018512, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, xref=4, ext=[AuthorCompanyExt(id=1218291754516795729, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754500018512, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4 College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, China), AuthorCompanyExt(id=1218291754520990035, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, companyId=1218291754500018512, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4 河南科技大学食品与生物工程学院, 河南 洛阳 471023)])], figs=[ArticleFig(id=1218291759084392994, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.1, caption=Physical appearance of antimicrobial peptide PMAP-24KK hydrogel

A-hydrogel with PMAP-24KK; B-hydrogel without PMAP-24KK.

, figureFileSmall=LzwX3QzJditUdJu95wePCg==, figureFileBig=EIgkFouv9GE13BX6M+R6PQ==, tableContent=null), ArticleFig(id=1218291759168279081, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图1, caption=猪源抗菌肽-24KK(PMAP-24KK)水凝胶的物理外观

A-含PMAP-24KK的水凝胶;B-不含PMAP-24KK的水凝胶。

, figureFileSmall=LzwX3QzJditUdJu95wePCg==, figureFileBig=EIgkFouv9GE13BX6M+R6PQ==, tableContent=null), ArticleFig(id=1218291759403160117, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.2, caption=Scanning electron microscopic observations of antimicrobial peptide PMAP-24KK hydrogel

A-hydrogel with PMAP-24KK; B-hydrogel without PMAP-24KK.

, figureFileSmall=MGQDk8y5ZRvWbuPMEp4uaw==, figureFileBig=t1NtuvPFKuymi0gqBRCljA==, tableContent=null), ArticleFig(id=1218291759503823419, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图2, caption=PMAP-24KK水凝胶的扫描电子显微镜观察图

A-含PMAP-24KK的水凝胶;B-不含PMAP-24KK的水凝胶。

, figureFileSmall=MGQDk8y5ZRvWbuPMEp4uaw==, figureFileBig=t1NtuvPFKuymi0gqBRCljA==, tableContent=null), ArticleFig(id=1218291759600292416, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.3, caption=Swelling rate(A), moisture content(B), and water retention rate(C) of antimicrobial peptide PMAP-24KK hydrogel. n=3,$\bar{x}±s$, figureFileSmall=1razn4LPoJ5G96G//znfeg==, figureFileBig=7Rh5xt7Sn1O3yVbDl3T+Ig==, tableContent=null), ArticleFig(id=1218291759679984195, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图3, caption=PMAP-24KK水凝胶的溶胀率(A)、含水率(B)和保水率(C)。n=3,$\bar{x}±s$, figureFileSmall=1razn4LPoJ5G96G//znfeg==, figureFileBig=7Rh5xt7Sn1O3yVbDl3T+Ig==, tableContent=null), ArticleFig(id=1218291759747093066, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.4, caption=Drug release rate of antimicrobial peptide PMAP-24KK hydrogel. n=3,$\bar{x}±s$, figureFileSmall=s9DwitMG2OsszYoq9SsLSQ==, figureFileBig=opo8nV5MVrhgDb4SNZe5qQ==, tableContent=null), ArticleFig(id=1218291759826784846, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图4, caption=PMAP-24KK水凝胶的药物释放率。n=3,$\bar{x}±s$, figureFileSmall=s9DwitMG2OsszYoq9SsLSQ==, figureFileBig=opo8nV5MVrhgDb4SNZe5qQ==, tableContent=null), ArticleFig(id=1218291759910670932, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.5, caption=Sustained release test results of antimicrobial peptide PMAP-24KK hydrogel

A-PMAP-24KK hydrogel at 12 h after preparation; B-PMAP-24KK hydrogel at 24 h after preparation; C-PMAP-24KK hydrogel at 36 h after preparation; D-the diameter of inhibition zone of PMAP-24KK sustained release from hydrogel at different time points.n=3,$\bar{x}±s$.

, figureFileSmall=AuP100Ac0qBzNFqQbc70Ew==, figureFileBig=Hk5GimCZzZNRWQPSUoBumw==, tableContent=null), ArticleFig(id=1218291760019722842, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图5, caption=PMAP-24KK水凝胶的缓释试验结果

A-制备12 h后的PMAP-24KK水凝胶;B-制备24 h后的PMAP-24KK水凝胶;C-制备36 h后的PMAP-24KK水凝胶;D-不同时间点抗菌肽水凝胶缓释PMAP-24K的抑菌圈直径。n=3,$\bar{x}±s$。

, figureFileSmall=AuP100Ac0qBzNFqQbc70Ew==, figureFileBig=Hk5GimCZzZNRWQPSUoBumw==, tableContent=null), ArticleFig(id=1218291760120386145, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.6, caption=Antimicrobial barrier test results of antimicrobial peptide PMAP-24KK hydrogel

A-Staphylococcus aureus; B-Listeria monocytogenes; C-Salmonella choleraesuis; D-Salmonella typhimurium; 1-the plate coated with PMAP-24KK hydrogel; 2-the plate coated without PMAP-24KK hydrogel

, figureFileSmall=jnAGB2GCare4dbrtqxCqVw==, figureFileBig=T730AornM8wNczVqwQO8pg==, tableContent=null), ArticleFig(id=1218291760216855141, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图6, caption=PMAP-24KK水凝胶的抗菌屏障试验结果

A-金黄色葡萄球菌;B-单增李斯特菌;C-猪霍乱沙门菌;D-鼠伤寒沙门菌;1-覆盖PMAP-24KK水凝胶的平板;2-不覆盖PMAP-24KK水凝胶的平板。

, figureFileSmall=jnAGB2GCare4dbrtqxCqVw==, figureFileBig=T730AornM8wNczVqwQO8pg==, tableContent=null), ArticleFig(id=1218291760304935531, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Fig.7, caption=Effects of antibacterial peptide PMAP-24KK hydrogel on skin wound healing in mice(×200)

A-healthy group; B-trauma group; C-hydrogel group; D-PMAP-24KK group; E-PMAP-24KK hydrogel group; F-antibiotic group.

, figureFileSmall=6bl2KaQPHFQMt6H6qkP0bQ==, figureFileBig=FZfYIqVZQSkitVVjuSNXng==, tableContent=null), ArticleFig(id=1218291760401404527, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=图7, caption=PMAP-24KK水凝胶对小鼠皮肤创口愈合的影响效果(×200)

A-健康组;B-创伤组;C-水凝胶组;D-PMAP-24KK组;E-PMAP-24KK水凝胶组;F-抗生素组。

, figureFileSmall=6bl2KaQPHFQMt6H6qkP0bQ==, figureFileBig=FZfYIqVZQSkitVVjuSNXng==, tableContent=null), ArticleFig(id=1218291760481096308, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=EN, label=Tab.1, caption=

Drug susceptibility test results of antimicrobial peptide PMAP-24KK hydrogel. n=3,$\bar{x}±s$

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains Bacteriostatic diameters/mm
Blank hydrogel PMAP-24KK hydrogel PMAP-24KK Ceftiofur sodium
Staphylococcus aureus ATCC 25923 0 33.00±1.001) 35.33±0.581) 24.00±5.29
Listeria monocytogenes 10403S 0 23.33±1.531) 24.67±1.531) 13.67±0.58
Salmonella choleraesuis C78-1 0 18.00±1.001)2) 24.33±0.581) 15.33±0.58
Salmonella typhimurium SL1344 0 18.33±0.581)2) 24.33±1.151) 14.33±1.53
), ArticleFig(id=1218291760548205174, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291751266210062, language=CN, label=表1, caption=

PMAP-24KK水凝胶的药敏试验结果。n=3,$\bar{x}±s$

, figureFileSmall=null, figureFileBig=null, tableContent=
Strains Bacteriostatic diameters/mm
Blank hydrogel PMAP-24KK hydrogel PMAP-24KK Ceftiofur sodium
Staphylococcus aureus ATCC 25923 0 33.00±1.001) 35.33±0.581) 24.00±5.29
Listeria monocytogenes 10403S 0 23.33±1.531) 24.67±1.531) 13.67±0.58
Salmonella choleraesuis C78-1 0 18.00±1.001)2) 24.33±0.581) 15.33±0.58
Salmonella typhimurium SL1344 0 18.33±0.581)2) 24.33±1.151) 14.33±1.53
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抗菌肽PMAP-24KK水凝胶的制备及抗菌活性研究
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毛付康 1, 2 , 范文琦 1, 2 , 路思阳 1, 2 , 王君辉 1, 2 , 王恩典 1, 2 , 崔靖靖 1, 2 , 王晓利 3 , 牛明福 4 , 廖成水 1, 2, *
中国药学杂志 | 论著 2024,59(13): 1219-1225
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中国药学杂志 | 论著 2024, 59(13): 1219-1225
抗菌肽PMAP-24KK水凝胶的制备及抗菌活性研究
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毛付康1, 2, 范文琦1, 2, 路思阳1, 2, 王君辉1, 2, 王恩典1, 2, 崔靖靖1, 2, 王晓利3, 牛明福4, 廖成水1, 2, *
作者信息
  • 1 河南科技大学功能微生物与畜禽健康实验室, 河南 洛阳 471023
  • 2 洛阳市活载体生物材料与动物疫病防控重点实验室, 河南 洛阳 471023
  • 3 河南科技大学基础医学与法医学院, 河南 洛阳 471023
  • 4 河南科技大学食品与生物工程学院, 河南 洛阳 471023

    • 毛付康,男,硕士研究生 研究方向:食源性微生物感染与新型药物开发研究

通讯作者:

* 廖成水,男,副教授,博士 研究方向:食源性微生物感染与新型药物开发研究 Tel:(0379)64563979
Preparation and Antibacterial Activity of Antimicrobial Peptide PMAP-24KK Hydrogel
Fukang MAO1, 2, Wenqi FAN1, 2, Siyang LU1, 2, Junhui WANG1, 2, Endian WANG1, 2, Jingjing CUI1, 2, Xiaoli WANG3, Mingfu NIU4, Chengshui LIAO1, 2, *
Affiliations
  • 1 Laboratory of Functional Microorganisms and Animal Health, Henan University of Science and Technology, Luoyang 471023, China
  • 2 Key Laboratory of Live Carrier Biomaterials and Animal Disease Prevention and Control in Luoyang, Luoyang 471023, China
  • 3 College of Basic Medicine and Forensic Medicine, Henan University of Science and Technology, Luoyang 471023, China
  • 4 College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, China
出版时间: 2024-07-08 doi: 10.11669/cpj.2024.13.006
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目的 制备含猪源抗菌肽-24KK(porcine myeloid antimicrobial peptide-24KK,PMAP-24KK)的水凝胶,评价其理化性能和抗菌活性。方法 将PMAP-24KK与海藻酸钠、碳酸钙和葡萄糖酸内酯结合得到PMAP-24KK水凝胶,首先分析PMAP-24KK水凝胶的形成时间、溶胀率、含水率、保水率和释放率等理化性质,然后进行药敏试验测定其对细菌的体外抗菌活性、缓释和抗菌屏障以及对小鼠皮肤创伤愈合的效果。结果 PMAP-24KK水凝胶在2 min左右可以形成具有网状结构的凝胶态,溶胀率和含水率均在10 h达到平衡状态,分别为(158.83±3.26)%和(61.01±2.41)%,24 h的保水率和释放率约为55%和81%。PMAP-24KK水凝胶对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌表现出抑菌效果。PMAP-24KK水凝胶18 h内的缓释作用没有明显区别,18 h后开始逐渐下降至36 h无抗菌活性。PMAP-24KK水凝胶能有效地起到抗菌屏障作用,且有助于小鼠创伤模型皮肤伤口的愈合。结论 本研究成功制备获得具有良好理化特性和抗菌活性的PMAP-24KK水凝胶,且具有较好的缓释和抗菌屏障以及促进伤口愈合的作用,为抗菌肽水凝胶应用的进一步研究奠定基础。

抗菌肽  /  猪源抗菌肽-24KK  /  海藻酸钠  /  水凝胶  /  抗菌活性

OBJECTIVE To prepare hydrogels containing antimicrobial peptide PMAP-24KK and evaluate antibacterial activity. METHODS The antimicrobial peptide PMAP-24KK was combined with sodium alginate, calcium carbonate, and gluconic acid-δ-lactone to form the PMAP-24KK hydrogel. Firstly, the formation time, swelling rate, moisture content, water retention rate, and release rate of PMAP-24KK hydrogel were analyzed. Then, the effect of PMAP-24KK hydrogel on the antibacterial activity in vitro, the sustained release, and the antibacterial barrier were determined by drug sensitivity test and skin wound healing in mice was analyzed. RESULTS The results showed that the PMAP-24KK hydrogel could form a gel state with network structure in about 2 min, and the swelling rate and moisture content reached equilibrium at 10 h, which were (158.83±3.26)% and (61.01±2.41)%, respectively. The water retention rate and release rate were about 55% and 81% after 24 h, respectively. The PMAP-24KK hydrogel showed the inhibitory effect on Staphylococcus aureus, Listeria monocytogenes, Salmonella choleraesuis, and Salmonella typhimurium. The sustained-release activity of PMAP-24KK hydrogel had no significant difference in 18 h, and gradually decreased after 18 h to 36 h without antibacterial activity. The PMAP-24KK hydrogel can effectively act as an antibacterial barrier and contribute to wound healing in mouse wound model. CONCLUSION Antimicrobial peptide PMAP-24KK hydrogel with good physicochemical properties and antibacterial activity is successfully prepared in this study, and it has good sustained-release and antibacterial barrier and promote wound healing, the study laid a foundation for further application research of antimicrobial peptide hydrogel.

antimicrobial peptide  /  porcine myeloid antimicrobial peptide-24KK  /  sodium alginate  /  hydrogel  /  antibacterial activity
毛付康, 范文琦, 路思阳, 王君辉, 王恩典, 崔靖靖, 王晓利, 牛明福, 廖成水. 抗菌肽PMAP-24KK水凝胶的制备及抗菌活性研究. 中国药学杂志, 2024 , 59 (13) : 1219 -1225 . DOI: 10.11669/cpj.2024.13.006
Fukang MAO, Wenqi FAN, Siyang LU, Junhui WANG, Endian WANG, Jingjing CUI, Xiaoli WANG, Mingfu NIU, Chengshui LIAO. Preparation and Antibacterial Activity of Antimicrobial Peptide PMAP-24KK Hydrogel[J]. Chinese Pharmaceutical Journal, 2024 , 59 (13) : 1219 -1225 . DOI: 10.11669/cpj.2024.13.006
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抗生素对细菌感染具有良好的预防和治疗效果,在医学领域具有举足轻重的地位。抗生素还具有群体防治和促进动物生长的作用,一些国家在饲料中添加远超规定用量的抗生素,导致动物性食品药物残留、环境污染等问题日益严重[1]。抗生素滥用使得微生物在进化过程中不断产生新的耐药机制,给传染病的防治和食品公共卫生安全带来巨大挑战[2]。因此,研发安全、高效和绿色的新型抗生素替代产品已迫在眉睫。
抗菌肽是一种具有抗菌作用的小分子蛋白质,通常由12~50个氨基酸组成,具有2~7个正电荷。抗菌肽具有抗菌性、热稳定性以及低耐药性和低残留量,被誉为“天然抗菌剂”[3]。抗菌肽的主要抗菌机制是破坏细菌的细胞膜,而细胞壁及其nty组分则是抗菌肽的主要靶标[4]。抗菌肽还具有抗真菌、抗病毒、抗寄生虫、免疫调节、抑制癌细胞等多种生物学作用[5]。猪源抗菌肽是从猪体内分离得到的一类抗菌肽,目前已发现16种具有不同螺旋结构的猪源抗菌肽。根据DNA结构和氨基酸排列特点将其分为半胱氨酸防御素超家族、Cathelicidins家族、Cecropins家族和NK-lysin家族4大类[6],其中猪髓源抗菌肽(porcine myloid antimicrobial peptide, PMAP)属于Cathelicidins家族抗菌肽[7]。
水凝胶是一种具有三维聚合物网络结构的材料,与血液、体液和动物性组织生物相容性很好,作为细胞外基质替代物广泛应用于组织工程、再生医学、药物输送和伤口敷料等方面[8]。同时,许多食品水凝胶也应用于食品工业领域[9]。抗菌肽易降解限制了其稳定性和抗菌效果,而将其与水凝胶结合后的局部给药可免于分解[10]。Liu等[11]截取PMAP-36的N端前24个氨基酸,并在C末端加入2个赖氨酸得到了一个新型抗菌肽PMAP-24KK,氨基酸序列为GRFRRLRKKTRKRLKKIGKVLKWIKK,PMAP-24KK具有良好抗菌活性。为提高抗菌肽稳定性和持续释放能力,最大限度发挥抗菌和促进伤口愈合作用,本研究将PMAP-24KK与海藻酸钠水凝胶结合制备PMAP-24KK水凝胶,然后测定其基本理化特性,最后评价PMAP-24KK水凝胶对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌4种食源性病原菌的抗菌活性,从而为进一步研制高效、稳定的抗菌肽水凝胶奠定基础。
1 材料
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1.1 菌株与试剂
金黄色葡萄球菌ATCC 25923、单增李斯特菌10403S、鼠伤寒沙门菌SL1344和猪霍乱沙门菌C78-1(河南科技大学功能微生物与畜禽健康实验室);酵母提取物、蛋白胨、胰蛋白胨大豆肉汤、胰蛋白胨大豆琼脂、脑心浸液肉汤和琼脂粉(青岛高科园海博生物技术有限公司);头孢噻呋钠(河南科技大学校医院);PMAP-24KK(上海科肽生物科技有限公司合成);海藻酸钠、碳酸钙、葡萄糖酸内酯、盐酸、氯化钾、氢氧化钠、氯化钠、磷酸氢二钠、磷酸二氢钠(国药集团化学试剂有限公司)。
1.2 主要仪器设备
超净操作台SW-CJ-2ED(苏州净化设备有限公司);全波段酶标仪Infinite M Nano(河南德泉兴业商贸有限公司);电热恒温培养箱HH-B11-BS-Ⅱ(上海跃进医疗器械有限公司);冰箱BCD-206TASJX(青岛海尔股份有限公司);电子天平JJ2002(上海浦春计量仪器有限公司);高温压力蒸汽灭菌锅LDZX-50KBS(上海申安医疗器械厂);200 μL和1 mL微量单道移液器(德国Eppendorf公司);气浴恒温振荡器CHA-SA(常州迅生仪器有限公司);扫描电子显微镜SU8100(日本Hitachi有限公司)。
2 方法
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2.1 PMAP-24KK水凝胶的制备与形态观察
称取适量PMAP-24KK冻干粉于3 mL无菌去离子水中,称取0.050 0 g海藻酸钠溶于3 mL抗菌肽溶液中得到1号液。称取0.101 0 g碳酸钙和0.071 2 g葡萄糖酸内酯分别于10 mL无菌去离子水中得到2号液和3号液,摇匀备用。取2号液和3号液各1 mL于1号液中使得抗菌肽终质量浓度为200 μg·mL-1,置于振荡器上快速振荡均匀,静置凝固后拍照观察PMAP-24KK水凝胶的物理形态。同时利用扫描电子显微镜观察PMAP-24KK水凝胶的内部微观结构。
2.2 PMAP-24KK水凝胶形成时间的测定
采用小瓶倒置试验测定凝胶形成时间,即在37 ℃下每10 s对倒置小瓶进行观察,混合液不倒流即表明已经凝胶化,记录PMAP-24KK水凝胶形成时间。
2.3 PMAP-24KK水凝胶溶胀率、含水率、保水率的测定
首先在室温下将PMAP-24KK水凝胶预先烘干水分,分析天平称量得到水凝胶的质量W0,然后置于去离子水中,每2 h取出水凝胶,用吸水纸去除表面水分后分析天平称量得到水凝胶的质量Wt,直至水凝胶的质量不发生变化,也就是达到溶胀平衡。根据下列公式计算水凝胶的溶胀率和含水率:溶胀率%=(Wt-W0)/W0×100%;含水率%=(Wt-W0)/Wt×100%。
为了测定水凝胶的保水率,将制备好的水凝胶置于去离子水中,使其达到溶胀平衡后取出,分析天平称量得到水凝胶的质量W0,再将达到溶胀平衡后的水凝胶置于室温,在35%湿度的条件下每2 h测定其质量Wt。根据公式计算水凝胶的保水率:保水率%=Wt/W0×100%。
2.4 PMAP-24KK水凝胶中抗菌肽的释放量和释放率的测定
将PMAP-24KK水凝胶置于30 mL去离子水溶液中,分别于不同时间点(2、4、6、8、12、14、24 h)用移液管收集3 mL水溶液,并补充等体积的去离子水溶液,保持溶液体积不变。采用紫外分光光度计在257 nm处测定样品中抗菌肽的浓度,以评价PMAP-24KK水凝胶中PMAP-24KK的释放量和释放率。
2.5 PMAP-24KK水凝胶的抗菌性能测定
为检测PMAP-24KK水凝胶对不同细菌的抗菌活性,通过纸片扩散法测定其对金黄色葡萄球菌ATCC 25923、单增李斯特菌10403S、猪霍乱沙门菌C78-1和鼠伤寒沙门菌SL1344的体外抗菌活性。将待测细菌培养至对数生长期,用PBS溶液将其调整为0.5麦氏浊度标准后均匀涂抹相应固体培养基上,然后将PMAP-24KK和头孢噻呋钠(10 μg)药敏片贴到琼脂平板上,37 ℃孵育18 h后测量抑菌圈的直径。
2.6 PMAP-24KK水凝胶的缓释抗菌效果测定
以金黄色葡萄球菌为测试菌评价水凝胶的缓释效果。将制备好的PMAP-24KK水凝胶放置于室温条件,每12 h将其贴至在涂布金黄色葡萄球菌菌液的固体培养基上,37 ℃恒温箱孵育18 h后测量其抑菌圈直径,从而评价抗菌肽水凝胶的药物缓释情况。
2.7 PMAP-24KK水凝胶的抗菌屏障效果测定
将制备好的PMAP-24KK水凝胶覆盖到固体培养基上,然后将金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌接种于水凝胶表面,以接种细菌且未覆盖水凝胶的固体培养基作为对照组。37 ℃恒温箱中孵育12 h后取出水凝胶,平板继续在37 ℃恒温箱培养18 h观察琼脂培养基上的细菌生长情况。
2.8 PMAP-24KK水凝胶的保质期测定
将制备好的PMAP-24KK水凝胶分别在室温下和4 ℃保存,按“2.5”项下药敏纸片法,以金黄色葡萄球菌为测试菌,每10 d进行抗菌肽PMAP-24KK水凝胶抗菌性能测定。
2.9 PMAP-24KK水凝胶对小鼠创伤效果评价
将小鼠随机分为6组:健康组、模型组、水凝胶组、PMAP-24KK组、含PMAP-24KK的水凝胶组和抗生素组。各组小鼠进行背部脱毛,并在健康组外的5组小鼠背部制作一个直径约1.5 cm的创口,将6.5×1010 CFU·mL-1的金黄色葡萄球菌悬浮液接种于创口上。感染后2 h开始治疗,每天治疗1次,持续10 d,最后采集小鼠伤口处的皮肤组织经HE染色进行组织病理学观察。
3 结果
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3.1 PMAP-24KK水凝胶的形态
海藻酸钠、碳酸钙和葡萄糖酸内酯制备得到的水凝胶形成均匀凝胶状,外观呈乳白色,质地均匀,形态稳定、表面光滑且富有弹性(图1)。扫描电子显微镜观察结果显示,PMAP-24KK水凝胶三维网络结构均匀,具有一定大小的孔洞,各孔洞结构之间相互连通,且孔径大小均匀,与不含抗菌肽水凝胶相比内部结构没有明显差别(图2)。表明PMAP-24KK对水凝胶的内部孔隙结构没有明显影响,依旧可以形成良好的三维孔状结构。
3.2 PMAP-24KK水凝胶的形成时间
小瓶倒置试验测定PMAP-24KK水凝胶形成时间,结果显示,37 ℃下PMAP-24KK水凝胶的平均凝胶化时间为(2.21±0.32) min。
3.3 PMAP-24KK水凝胶的溶胀率、含水率和保水率
将PMAP-24KK水凝胶放置于去离子水中,每2 h取出称量水凝胶的质量以计算水凝胶的溶胀速率,结果见图3A,PMAP-24KK水凝胶在前6 h迅速吸水膨胀,吸水率最快,随后吸水速率逐渐趋于平缓,在10 h达到溶胀平衡。PMAP-24KK水凝胶在不同时间点(0、2、4、6、8、12、24 h)的含水率见图3B,在0~10 h含水率逐渐提高,在10 h以后趋于平缓,达到相对平衡状态。最后,将PMAP-24KK水凝胶样品放置于去离子水中,使其达到溶胀平衡后放置于室温,每2 h测定其质量,随着时间延长PMAP-24KK水凝胶的保水率呈逐渐下降趋势,24 h时保水率约为50%(图3C)。表明PMAP-24KK水凝胶具有较好的吸水和保湿能力。
3.4 PMAP-24KK水凝胶中抗菌肽的释放量和释放率
水凝胶的释放率是评价水凝胶性能的一个重要指标。将PMAP-24KK水凝胶置于30 mL去离子水溶液中,于不同时间点(2、4、6、8、12、14、24 h)采用紫外分光光度计在257 nm处测定样品的吸光度值。结果见图4,PMAP-24KK水凝胶12 h前能快速释放PMAP-24KK,其释放率上升趋势明显,在12 h以后趋于平缓,在75%左右,且24 h时PMAP-24KK的释放率达到80.28%。表明PMAP-24KK与海藻酸钠水凝胶结合后能稳定释放出PMAP-24KK。
3.5 PMAP-24KK水凝胶的抗菌性能测定结果
采用药敏纸片法测定PMAP-24KK、PMAP-24KK水凝胶对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌的抑菌圈直径,结果见表1。空白水凝胶没有任何抑菌活性,PMAP-24KK对4种测试菌株的抑菌圈直径与头孢噻呋钠对照相比具有显著差异(P<0.05),PMAP-24KK水凝胶对4种菌株也表现出相似的抑制效果,与头孢噻呋钠相比具有显著差异(P<0.05)。与PMAP-24KK相比,PMAP-24KK水凝胶对金黄色葡萄球菌和单增李斯特菌的抗菌活性没有发生明显变化,但对猪霍乱沙门菌和鼠伤寒沙门菌的抗菌活性有所降低(P<0.05)。表明PMAP-24KK与海藻酸钠水凝胶结合后不影响其抗菌活性。
3.6 PMAP-24KK水凝胶的缓释抗菌效果测定结果
为进一步观察PMAP-24KK水凝胶缓释药物的抗菌效果,将PMAP-24KK水凝胶于室温每6 h检测其抗菌活性,结果见图5。PMAP-24KK水凝胶放置12 h后对金黄色葡萄球菌具有抑菌作用(图5A),与新鲜制备的PMAP-24KK水凝胶抑菌能力相当,24 h时抗菌活性下降将近一半(图5B、D),到36 h时PMAP-24KK水凝胶几乎无抑菌圈(图5C),整体上12 h前释放PMAP-24KK的抗菌能力无变化,18 h开始逐渐下降(图5D)。表明PMAP-24KK水凝胶具有良好的缓释效果。
3.7 PMAP-24KK水凝胶的抗菌屏障效果测定结果
为观察PMAP-24KK水凝胶抗菌的屏障作用,在固体培养基上面覆盖一层水凝胶,将金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌菌液分别接种于水凝胶表面,以接种细菌未覆盖水凝胶的培养基作为对照组,结果见图6。当揭下水凝胶被时,固体琼脂没有损坏,这说明水凝胶不会由于从伤口上去除敷料而造成二次伤害。与对照组不同的是,覆盖PMAP-24KK水凝胶的琼脂平板上,12 h时没有观察到金黄色葡萄球菌生长(图6)。表明PMAP-24KK水凝胶有效地对细菌起到屏障作用。
3.8 PMAP-24KK水凝胶的保质期测定结果
药敏试验结果显示,无论是常温保存还是4 ℃保存,PMAP-24KK水凝胶随着储存时间延长抗菌活性逐渐降低,储藏60 d时仍能表现出对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌的抑菌能力。同时,相同保存时间下,PMAP-24KK水凝胶常温保存时的抗菌活性比4 ℃保存时差。表明PMAP-24KK水凝胶能够储藏一定时间。
3.9 PMAP-24KK水凝胶的体内治疗效果评价
将PMAP-24KK水凝胶用于小鼠创口,第10天时PMAP-24KK水凝胶组小鼠的创口基本愈合,采集小鼠伤口愈合处的皮肤组织进行组织病理学观察。HE染色结果显示,健康空白组小鼠毛囊分布均匀,但创伤组小鼠成纤维细胞和炎性细胞较多,仅有少量毛囊分布(图7)。PMAP-24KK组和水凝胶组效果不佳,小鼠分化程度低,有成纤维细胞分布和炎性细胞数量减少。PMAP-24KK水凝胶组的小鼠毛囊密集且分化程度较高,炎性细胞大量减少。抗生素组也有较多毛囊分布但分化程度不高,有少量炎性细胞浸润。表明PMAP-24KK水凝胶作为创伤敷料可促进创口愈合。
4 讨论
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将多糖、乙二醇等亲水高分子与多种杀菌剂相结合可形成抗菌水凝胶[12]。抗菌水凝胶可持续地在受感染的位置释放抗菌药,且具有良好的组织黏附性,有助于组织修复[13],也可作为抗菌涂层有效保护医疗器械或植入设备。海藻酸是一种由α-L-古罗糖醛酸(G)和β-D-甘露糖醛酸(M)2种结构单元通过1,4-糖苷键连接而成的褐藻多糖[14]。海藻酸钠是美国食品药品监督管理局认定的GRAS钠盐,可用于食品和医用生物材料,海藻酸钠水凝胶是一种具有丰富水分的三维网状结构[15],其结构与细胞外基质相似,吸收大量的水分膨胀后仍能维持原来的结构。
抗菌肽和水凝胶经过物理、化学方法结合后可以改善抗菌肽的稳定性和靶向性[16]。本研究将前期研究证实具有良好抗菌功能的抗菌肽PMAP-24KK与海藻酸钠水凝胶相结合,PMAP-24KK可进入水凝胶内部网状结构中。水凝胶的溶胀率和含水率、药物释放率都有着重要联系,水凝胶的溶胀率越高,其含水率和释放率也越高[17],本研究得到的抗菌肽PMAP-24KK水凝胶溶胀率和含水率可达到160.73%和61.65%。对液体的吸附及保湿性能是衡量伤口敷料作用的重要指标,水凝胶是一种亲水性的网状聚合物溶胀剂,它可以吸收和保留大部分的水分,但不会溶于水,PMAP-24KK水凝胶保水率在12 h仍可达到55%,这表明海藻酸钠水凝胶含水量很高。普通纱布不具有保水性能,保水率高的水凝胶可以提供湿润环境,促进伤口愈合。
与水凝胶相结合的抗菌肽可以成功从水凝胶中释放出来,释放率在24 h可达到80.28%,PMAP-24KK水凝胶对金黄色葡萄球菌、单增李斯特菌、猪霍乱沙门菌和鼠伤寒沙门菌的生长具有抑制效果,并且与PMAP-24KK相比并没有显著降低其广谱高效的抗菌活性。药物缓释在临床应用中具有重要意义[18],传统的给药方式可能导致药物在体内的浓度不稳定,而药物缓释可以提高药物的利用率和药效,既减少给药次数,又使药物浓度得到稳定[19]。本研究发现抗菌肽PMAP-24KK水凝胶能持续释放抗菌肽,36 h内对金黄色葡萄球菌均有抑制作用,表明制备的抗菌肽水凝胶具有良好的缓释效果,可以提高其在临床应用时的利用率,减少给药次数,且相对于抗菌肽单独使用更加稳定。
屏障保护是保护伤口和周围伤口的有效方法。当出现较大伤口时使用的敷料应尽快形成保护膜,防止外界细菌感染,因此要求水凝胶具有抗菌屏障作用[20]。PMAP-24KK水凝胶能有效地对金黄色葡萄球菌起到至少12 h的屏障作用,在临床应用过程中可以防止细菌的进一步感染。伤口的愈合需要很长一段时间,水凝胶的亲和力会降低药物对身体的刺激性,而水凝胶则能有效地防止其损伤,使其分子活性维持更久[21]。进一步的小鼠创伤模型也证实了PMAP-24KK水凝胶可以降低伤口的炎症,有助于伤口愈合。
综上所述,本研究成功制备了性能优良的抗菌肽PMAP-24KK水凝胶,可缓慢释放具有抗菌活性的抗菌肽PMAP-24KK,且具有较好抗菌屏障和促进伤口愈合作用,在生命科学和食品工业中具有一定的应用潜力。
基金
收起
  • 河南省科技攻关项目(182102110061)
  • 河南科技大学青年骨干教师培养计划(13450009)
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2024年第59卷第13期
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doi: 10.11669/cpj.2024.13.006
  • 接收时间：2023-01-12
  • 首发时间：2026-01-14
  • 出版时间：2024-07-08
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  • 收稿日期：2023-01-12
基金
河南省科技攻关项目(182102110061)
河南科技大学青年骨干教师培养计划(13450009)
作者信息
    1 河南科技大学功能微生物与畜禽健康实验室, 河南 洛阳 471023
    2 洛阳市活载体生物材料与动物疫病防控重点实验室, 河南 洛阳 471023
    3 河南科技大学基础医学与法医学院, 河南 洛阳 471023
    4 河南科技大学食品与生物工程学院, 河南 洛阳 471023

通讯作者:

* 廖成水,男,副教授,博士 研究方向:食源性微生物感染与新型药物开发研究 Tel:(0379)64563979
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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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