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Article(id=1198656145889001842, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0062, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1673971200000, receivedDateStr=2023-01-18, revisedDate=1684771200000, revisedDateStr=2023-05-23, acceptedDate=null, acceptedDateStr=null, onlineDate=1763711495134, onlineDateStr=2025-11-21, pubDate=1694448000000, pubDateStr=2023-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763711495134, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763711495134, creator=13701087609, updateTime=1763711495134, updator=13701087609, issue=Issue{id=1198656143976399200, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='9', pageStart='2541', pageEnd='2834', issueExtLink='null', onlineDate='null', pubDate='1694448000000', pubDateStr='2023-09-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763711494677, creator='13701087609', updateTime=1763711620095, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1198656670072144034, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198656670072144035, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2794, endPage=2801, ext={EN=ArticleExt(id=1198656146232934777, articleId=1198656145889001842, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Hollow copper sulfide nanoparticles loading deferoxamine for photothermal antibacterial therapy and promoting angiogenesis, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Diabetic ulcer is recognized as a chronic nonhealing wound, often associated with bacterial infection and tissue necrosis, which seriously affect patients' health and quality of life. The traditional treatment methods exist some problems, such as bacterial resistance and secondary trauma, so it is urgent to find new methods to meet the requirements of diabetic ulcer treatment. In this study, we prepared a drug delivery system (DFO@CuS nanoparticles) based on hollow copper sulfide (CuS) nanoparticles loaded with deferoxamine (DFO), which realized the synergistic therapy of promoting angiogenesis and photothermal antibacterial. The morphological structure and particle size distribution of DFO@CuS nanoparticles were characterized by transmission electron microscopy and particle size analyzer, respectively. The antibacterial effect of DFO@CuS nanoparticles was evaluated by the plate coating method. The effects of DFO@CuS nanoparticles on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8 (cell counting kit-8) assay, cell scratch assay, and tube formation assay. The results showed that DFO@CuS nanoparticles were hollow and spherical in shape with an average particle size of (200.9 ±8.6) nm. DFO@CuS nanoparticles could effectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) under near-infrared (NIR) light irradiation. DFO@CuS nanoparticles showed negligible cytotoxicity and effective acceleration of cell migration and tube formation in a certain concentration range. In conclusion, the prepared DFO@CuS nanoparticles exhibit good photothermal antibacterial properties and pro-angiogenic effects, providing a basis for their application in the treatment of diabetic ulcer.

, authors=null, authorsList=Yi QIAO, Chun ZHANG, Yan-e MA, Jia-ling CHEN, Hai-jun SHEN, authorCompany=null, correspAuthors=Hai-jun SHEN, 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=1198656147738690002, articleId=1198656145889001842, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=中空硫化铜纳米粒负载去铁胺在光热抗菌及促血管生成方面的研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

糖尿病溃疡是一种难愈合性伤口, 常伴有细菌感染、组织坏死等情况, 严重影响患者生命健康与生活质量。传统的治疗方法存在细菌耐药、二次创伤等问题, 因此急需寻找新的方法以满足治疗需求。本研究制备了一种基于中空硫化铜(copper sulfide, CuS)纳米粒负载去铁胺(deferoxamine, DFO)的给药系统(DFO@CuS纳米粒), 实现了促进血管再生与光热抗菌的协同治疗。采用透射电镜、粒径分析仪等仪器对DFO@CuS纳米粒的形态结构、粒径分布等进行了表征, 利用平板涂布法评价了DFO@CuS纳米粒的抗菌效果, 通过CCK-8(cell counting kit-8)、细胞划痕、小管形成等实验评价了DFO@CuS纳米粒对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)增殖、迁移及成管能力的影响。研究结果表明: DFO@CuS纳米粒在透射电镜下呈中空球形, 平均粒径在(200.9 ±8.6) nm; DFO@CuS纳米粒在近红外光照射下可有效抑制耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌的生长; DFO@CuS纳米粒具有较低的细胞毒性, 在一定浓度范围内可有效促进细胞的迁移与成管。综上所述, 制备的DFO@CuS纳米粒具有良好的光热抗菌性能与促血管生成效应, 为其用于治疗慢性糖尿病溃疡提供了研究基础。

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*沈海俊, E-mail:
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Transmission electron microscope images of (A) CuS nanoparticles (CuS NPs) and (B) DFO@CuS nanoparticles. Scale bar = 50 nm. (C) The dynamic light scattering results of CuS NPs and DFO@CuS NPs. (D) Zeta potentials of deferoxamine (DFO), CuS NPs, and DFO@CuS NPs. (E) UV-vis spectra of DFO, CuS NPs, and DFO@CuS NPs , figureFileSmall=RJtSY/uaTIhrTEP5hhI9pw==, figureFileBig=UKcMC17zmMIZzmMM4xCBNg==, tableContent=null), ArticleFig(id=1198960233021273076, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=EN, label=null, caption=null, figureFileSmall=/G8ffk3DXNO1A8mFnqfbIg==, figureFileBig=WtiviD4TvfT53dCKYafaSg==, tableContent=null), ArticleFig(id=1198960233155489794, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=CN, label=Figure 2, caption= Photothermal effect of DFO@CuS NPs. A: Photothermal activity comparison of CuS NPs, DFO@CuS NPs, and water under 2.0 W·cm<sup>-2</sup> 980 nm near-infrared (NIR) laser irradiation for 5 min; B: Infrared thermal imaging of water and DFO@CuS NPs under 2.0 W·cm<sup>-2</sup> 980 nm NIR laser irradiation for 0-5 min; C: Photothermal conversion characterizations of DFO@CuS NPs with various concentrations under 2.0 W·cm<sup>-2</sup> 980 nm NIR laser irradiation for 5 min; D: The photothermal conversion cycling test of DFO@CuS NPs under 2.0 W·cm<sup>-2</sup> 980 nm NIR laser irradiation , figureFileSmall=/G8ffk3DXNO1A8mFnqfbIg==, figureFileBig=WtiviD4TvfT53dCKYafaSg==, tableContent=null), ArticleFig(id=1198960233260347406, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=EN, label=null, caption=null, figureFileSmall=MV6/0V02pyQot8qwc2lhEA==, figureFileBig=Rb7c3vhZ7uw7R01S7u+oPQ==, tableContent=null), ArticleFig(id=1198960233386176538, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=CN, label=Figure 3, caption= Antibacterial activity of DFO@CuS NPs at different concentrations (with or without NIR) <i>in vitro</i>. A: Photographs of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) bacterial colonies formed on Luria-Bertani (LB) agar plates with DFO@CuS NPs at different concentrations (with or without NIR); B: The corresponding colony forming units (CFU) count of MRSA with various treatments; C: Photographs of <i>Pseudomonas aeruginosa</i> (PA) bacterial colonies formed on LB agar plates with DFO@CuS NPs at different concentrations (with or without NIR); D: The corresponding CFU count of PA with various treatments. The power density was 2.0 W·cm<sup>-2</sup> for 5 min (<i>n</i> = 3, <span class="mag-xml-inline-formula">$ \stackrel{-}{x} $</span> ± <i>s</i>. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01, <sup>***</sup><i>P</i> < 0.001, <sup>****</sup><i>P</i> < 0.000 1) , figureFileSmall=MV6/0V02pyQot8qwc2lhEA==, figureFileBig=Rb7c3vhZ7uw7R01S7u+oPQ==, tableContent=null), ArticleFig(id=1198960233541365799, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=EN, label=null, caption=null, figureFileSmall=TItipVzxaTYnVwxrppViMg==, figureFileBig=cWqCXfRrLodGyXE8M5mw/w==, tableContent=null), ArticleFig(id=1198960233667194931, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1198656145889001842, language=CN, label=Figure 4, caption= <i>In vitro</i> cell viability, scratch assay, and tubule formation assay. A: Cell viability of human umbilical vein endothelial cells (HUVECs) treated with DFO@CuS NPs at different concentrations for 24 h; B: Digital images of HUVECs cells after treatment with DFO@CuS NPs at different concentrations for 30 h, Scale bar = 100 μm; C: <i>In vitro</i> tube formation of HUVECs after treatment with DFO@CuS NPs at different concentrations for 24 h. Scale bar = 200 μm; D: Quantification of tubule nodes and length by ImageJ (<i>n</i> = 3, <span class="mag-xml-inline-formula">$ \stackrel{-}{x} $</span> ± <i>s</i>. <sup>*</sup><i>P</i> < 0.05, <sup>****</sup><i>P</i> < 0.000 1 <i>vs</i> 0 µg·mL<sup>-1</sup> DFO@CuS NPs) , figureFileSmall=TItipVzxaTYnVwxrppViMg==, figureFileBig=cWqCXfRrLodGyXE8M5mw/w==, tableContent=null)], attaches=null, journal=Journal(id=1189982048455397383, delFlag=0, nameCn=药学学报, nameEn=Acta Pharmaceutica Sinica, nameHistory1=null, nameHistory2=null, issn=0513-4870, eissn=null, cn=11-2163/R, coden=null, periodic=0, language=CN, oaType=null, ccby=null, superviseOffice=null, ownerOffice=null, pubOffice=null, editorOffice=null, officeType=null, aims=null, clcCode=null, officeProv=null, officeCity=null, officeAddr=null, officeZip=null, officeEmail=null, officePhone=null, editDirector=null, officeDirector=null, officeDirectorPhone=null, officeStaffNum=null, officeEmpNum=null, coverPicUrl=BTxjudbJDVO4PqdBR6On6Q==, 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中空硫化铜纳米粒负载去铁胺在光热抗菌及促血管生成方面的研究
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乔屹 , 张纯 , 马燕娥 , 陈嘉玲 , 沈海俊 *
药学学报 | 研究论文 2023,58(9): 2794-2801
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药学学报 |研究论文 2023 , 58 (9) : 2794 -2801
中空硫化铜纳米粒负载去铁胺在光热抗菌及促血管生成方面的研究
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乔屹, 张纯, 马燕娥, 陈嘉玲, 沈海俊*
作者信息
  • 江苏大学医学院, 江苏 镇江 212013
通讯作者:
*沈海俊, E-mail:
Hollow copper sulfide nanoparticles loading deferoxamine for photothermal antibacterial therapy and promoting angiogenesis
Yi QIAO, Chun ZHANG, Yan-e MA, Jia-ling CHEN, Hai-jun SHEN*
Affiliations
  • School of Medicine, Jiangsu University, Zhenjiang 212013, China
出版时间: 2023-09-12 doi: 10.16438/j.0513-4870.2023-0062
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摘要
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糖尿病溃疡是一种难愈合性伤口, 常伴有细菌感染、组织坏死等情况, 严重影响患者生命健康与生活质量。传统的治疗方法存在细菌耐药、二次创伤等问题, 因此急需寻找新的方法以满足治疗需求。本研究制备了一种基于中空硫化铜(copper sulfide, CuS)纳米粒负载去铁胺(deferoxamine, DFO)的给药系统(DFO@CuS纳米粒), 实现了促进血管再生与光热抗菌的协同治疗。采用透射电镜、粒径分析仪等仪器对DFO@CuS纳米粒的形态结构、粒径分布等进行了表征, 利用平板涂布法评价了DFO@CuS纳米粒的抗菌效果, 通过CCK-8(cell counting kit-8)、细胞划痕、小管形成等实验评价了DFO@CuS纳米粒对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)增殖、迁移及成管能力的影响。研究结果表明: DFO@CuS纳米粒在透射电镜下呈中空球形, 平均粒径在(200.9 ±8.6) nm; DFO@CuS纳米粒在近红外光照射下可有效抑制耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌的生长; DFO@CuS纳米粒具有较低的细胞毒性, 在一定浓度范围内可有效促进细胞的迁移与成管。综上所述, 制备的DFO@CuS纳米粒具有良好的光热抗菌性能与促血管生成效应, 为其用于治疗慢性糖尿病溃疡提供了研究基础。

糖尿病溃疡  /  伤口愈合  /  光热治疗  /  抗菌  /  血管生成

Diabetic ulcer is recognized as a chronic nonhealing wound, often associated with bacterial infection and tissue necrosis, which seriously affect patients' health and quality of life. The traditional treatment methods exist some problems, such as bacterial resistance and secondary trauma, so it is urgent to find new methods to meet the requirements of diabetic ulcer treatment. In this study, we prepared a drug delivery system (DFO@CuS nanoparticles) based on hollow copper sulfide (CuS) nanoparticles loaded with deferoxamine (DFO), which realized the synergistic therapy of promoting angiogenesis and photothermal antibacterial. The morphological structure and particle size distribution of DFO@CuS nanoparticles were characterized by transmission electron microscopy and particle size analyzer, respectively. The antibacterial effect of DFO@CuS nanoparticles was evaluated by the plate coating method. The effects of DFO@CuS nanoparticles on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8 (cell counting kit-8) assay, cell scratch assay, and tube formation assay. The results showed that DFO@CuS nanoparticles were hollow and spherical in shape with an average particle size of (200.9 ±8.6) nm. DFO@CuS nanoparticles could effectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) under near-infrared (NIR) light irradiation. DFO@CuS nanoparticles showed negligible cytotoxicity and effective acceleration of cell migration and tube formation in a certain concentration range. In conclusion, the prepared DFO@CuS nanoparticles exhibit good photothermal antibacterial properties and pro-angiogenic effects, providing a basis for their application in the treatment of diabetic ulcer.

diabetic ulcer  /  wound healing  /  photothermal therapy  /  antibacterial  /  angiogenesis
乔屹, 张纯, 马燕娥, 陈嘉玲, 沈海俊. 中空硫化铜纳米粒负载去铁胺在光热抗菌及促血管生成方面的研究. 药学学报, 2023 , 58 (9) : 2794 -2801 . DOI: 10.16438/j.0513-4870.2023-0062
Yi QIAO, Chun ZHANG, Yan-e MA, Jia-ling CHEN, Hai-jun SHEN. Hollow copper sulfide nanoparticles loading deferoxamine for photothermal antibacterial therapy and promoting angiogenesis[J]. Acta Pharmaceutica Sinica, 2023 , 58 (9) : 2794 -2801 . DOI: 10.16438/j.0513-4870.2023-0062
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随着生活水平的不断提高, 糖尿病患者的数量也显著增长, 预计到2045年, 全球糖尿病患病人数将增加到6.29亿[1]。随之而来的是大量糖尿病并发症的发生, 其中最常见的就是糖尿病溃疡, 它们严重影响到了人们的健康, 甚至生命质量[2]。慢性糖尿病溃疡难以愈合的主要原因是由于新生血管受损和持续的细菌感染。血管生成缺乏会导致局部营养、氧气供应不足, 以及代谢产物的堆积会进一步加剧因细菌感染产生的炎症反应, 导致缺血、组织坏死等情况, 不利于伤口修复[3-5]。目前临床上常用的治疗方法为外科手术切除、高压氧疗、负压引流来控制病情, 但存在一定局限性, 而且长期使用抗生素会导致细菌耐药性的产生[6]。因此如何抑制创面上细菌的生长和促进血管新生成为目前研究的热点。
近些年, 光热治疗作为一种新兴疗法越来越受到研究者的关注[7-9], 相比于抗生素, 光热剂可以通过近红外光照射产生的局部高温直接破坏病原体的结构, 从而广谱地抑制细菌的活性, 减少耐药性的产生[10-12]。此外有研究显示, 光热疗法可以通过灭活细菌的活性物质, 破坏生物膜的形成, 促进药物的透皮效果[13-15]。硫化铜(copper sulfide, CuS) 因其优异的光热转换性能、简单的制备过程以及良好的生物相容性更是被广泛研究[16-18]。此外, 通过优化制备流程合成的中空CuS纳米粒具有比表面积大等特点, 是良好的药物载体, 被广泛地应用在与肿瘤相关疾病的治疗上[19-21]。受此启发, 本课题组设想可以利用CuS负载促血管生成药物来达到协同治疗的目的。
去铁胺(deferoxamine, DFO) 是临床上常用的铁离子螯合剂, 广泛运用于治疗慢性铁负载过重、急性铁中毒、脑出血等疾病当中[22-24]。近些年来, 大量研究表明DFO具有促进伤口愈合功能[25, 26]。缺氧诱导因子-1α (hypoxia inducible factor-1α, HIF-1α) 是一种转录因子, 可在细胞核内结合缺氧反应元件, 然后通过结合共激活因子p300, 上调血管生成相关基因的表达, 如血管内皮生长因子(vascular endothelial growth factor, VEGF)、成纤维细胞生长因子(fibroblast growth factor, FGF) 等, 从而促进血管再生[27, 28]。然而HIF-1α在常氧条件下会迅速被脯氨酸羟化酶(prolyl hydroxylase, PHD) 羟基化, 并通过泛素‒蛋白酶体途径降解。Fe2+是PHD的辅因子, 是其催化活性所必需的金属离子。DFO通过与Fe2+结合, 竞争性抑制PHD的活性, 从而稳定HIF-1α的表达[29], 继而促进血管生成相关蛋白的分泌, 加速血管新生。
综上所述, 本研究拟制备一种具有优良光热效应和促血管生成能力的新型给药系统(DFO@CuS纳米粒), 探究纳米粒对耐甲氧西林金葡菌(methicillin-resistant Staphylococcus aureus, MRSA)、铜绿假单胞菌(Pseudomonas aeruginosa, PA) 的抗菌效果, 并采用人脐静脉内皮细胞系(human umbilical vein endothelial cells, HUVECs) 作为模型细胞, 评价DFO@CuS纳米粒对HUVECs的促血管生成效果, 为DFO@CuS纳米粒在慢性糖尿病溃疡治疗上的应用提供实验依据。
材料与方法
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试剂与药品  聚乙烯吡咯烷酮(PVP-K30, 北京索莱宝科技有限公司, 1210J031); 无水氯化铜(G1816042)、九水合硫化钠(C2224294) (阿拉丁试剂有限公司); 水合肼(上海国药集团化学试剂有限公司, 20210830); 甲磺酸去铁胺(美国Sigma公司, BCCF5718); DMEM培养基(MA0212-Apr-13I)、磷酸盐缓冲液(PBS, MA0015-Feb-081)、青霉素-链霉素(23041546)、胰蛋白酶(22334415)、CCK-8试剂盒(苏州美仑生物科技有限公司, MA0218-2-Oct-19H); 胎牛血清(FBS, 依科赛生物科技有限公司, 12B215); Matrigel基质胶(美国BD公司, 1039001)。
主要仪器  纳米粒径电位分析仪(Zetasizer Nano ZS90, 英国Malvern Instrument公司); 透射电子显微镜(HT-7800, 日本日立公司); 紫外分光光度计(TU-1810, 北京普析通用仪器有限责任公司); 红外热成像仪(HT-19, 鑫泰仪器仪表有限公司); 980 nm激光仪(LWIRPD-5F, 北京镭志威光电技术有限公司); 倒置显微镜(CKX41, 日本Olympus公司); 酶标仪(800TS, 美国Bio-Tek公司); 血管生成载玻片(81506, 德国Ibidi公司)。
细胞培养  人脐静脉内皮细胞系(HUVECs) 购自中国科学院上海细胞库, 用含100 μg·mL-1链霉素、100 μg·mL-1青霉素、10%胎牛血清的DMEM高糖培养基培养于37 ℃含5% CO2的细胞培养箱中; 用0.25%胰蛋白酶消化传代; 细胞选用3~6代进行体外实验。
CuS纳米粒合成及表征  称量240 mg的PVP-K30溶于25 mL蒸馏水中, 混匀, 加入100 μL的浓度为0.5 mol·L-1的CuCl2溶液, 搅拌5 min。然后向混合液中依次加入25 mL pH = 9的NaOH溶液和8 μL的水合肼, 混合液立即变成棕黄色。继续搅拌5 min后加入200 μL (320 mg·mL-1) 的Na2S溶液。将反应液移至圆底烧瓶中, 60 ℃油浴搅拌加热2 h, 12 000 r·min-1离心15 min, 去上清, 蒸馏水重复洗涤2次, 得到空心CuS纳米粒, 最后分散在去离子水中保存。粒径电位分析仪测量CuS纳米粒的平均粒径与Zeta电位。透射电子显微镜观察CuS纳米粒的形态结构。紫外分光光度计扫描CuS纳米粒在200~1 100 nm处的吸收光谱。
DFO@CuS纳米粒合成及表征  将1 mL DFO溶液(1 000 µg·mL-1) 与400 µg CuS纳米粒混合, 室温搅拌24 h。将上述混合液离心洗涤以除去未负载的DFO, 得到DFO@CuS纳米粒, 将收集的上清液与3 mmol·L-1 FeCl3溶液等体积混匀, 反应30 min后, 使用紫外分光光度计测量490 nm处的吸光度, 通过DFO标准曲线法计算上清液中游离DFO的含量, 进而计算DFO@CuS纳米粒的包封率与载药量。粒径电位分析仪测量DFO@CuS纳米粒的平均粒径与Zeta电位。透射电子显微镜观察DFO@CuS纳米粒的形态结构。紫外分光光度计扫描DFO@CuS纳米粒在200~1 100 nm处的吸收光谱。
DFO@CuS纳米粒光热性能检测  将H2O、CuS纳米粒和DFO@CuS纳米粒溶液置于离心管中, 使用2 W·cm-2的近红外光(980 nm) 垂直照射, 红外热成像仪每隔30 s记录温度变化, 共计5 min。然后将不同浓度(0、50、100、200和400 μg·mL-1) 的DFO@CuS纳米粒溶液置于离心管中, 使用2 W·cm-2的近红外光(980 nm) 垂直照射5 min, 并通过红外热成像仪记录温度变化曲线。此外, 将样品进行多次激光“开-关”的循环照射以评价DFO@CuS纳米粒的光热稳定性。
DFO@CuS纳米粒抗菌性能检测  挑取单个菌落(耐甲氧西林金葡菌、铜绿假单胞菌) 至LB培养液, 在37 ℃摇床中活化至对数生长期, 将活化后的菌悬液稀释到1×106 CFU·mL-1, 吸取稀释后菌悬液与等体积不同浓度(0、50、100、200和400 μg·mL-1) 的DFO@CuS纳米粒溶液混匀。使用2 W·cm-2的近红外光(980 nm) 照射5 min, 以不光照组为对照。然后将处理后的菌悬液再次稀释至103 CFU·mL-1, 并取50 μL均匀涂板, 置于37 ℃培养过夜, 第二天拍照计数。本实验重复三次。
DFO@CuS纳米粒的细胞毒性测试  将HUVECs细胞以5×103/孔的密度接种于96孔板中, 37 ℃培养24 h后, 每孔加入100 µL含有不同浓度DFO@CuS纳米粒的完全培养基继续培养24 h。弃去含药培养液, 用PBS洗涤3次后, 每孔加入100 µL含有10% CCK-8试剂的完全培养液, 置于37 ℃培养箱继续孵育2 h, 用酶标仪检测450 nm波长下的吸光度。本实验重复三次。
细胞划痕实验  将HUVECs接种于6孔板中, 待其完全铺满培养板底时, 使用10 μL的枪头垂直地在细胞层上划出一条笔直的线。弃去各孔中的培养液, 并用PBS清洗3次, 以去除划下的细胞残渣与碎片。各孔加入含有2 mL不同浓度DFO@CuS纳米粒的低血清培养液(FBS ≤ 2%), 置于倒置显微镜下拍照后将6孔板放入培养箱中继续培养30 h。30 h后取出6孔板, 在倒置光学显微镜下观察并拍摄各组细胞划痕状态。
小管形成实验  将Matrigel基质胶在4 ℃下过夜解冻, 然后加入预冷的血管生成载玻片孔后, 放入37 ℃培养箱孵育30 min, 使其成胶。然后将与不同浓度DFO@CuS纳米粒共培养24 h的HUVECs收集重悬, 以每孔1×104的密度接种到血管生成载玻片中。37 ℃培养3 h后, 使用倒置显微镜观察细胞成管情况, 并使用Image J血管生成插件对其管长度和节点数进行测量记录。实验进行三次重复。
统计学方法  数据以$ \stackrel{-}{x} $ ± s表示, 采用GraphPad Prism 8.0统计软件对数据进行分析, 多组间均数比较采用单因素方差分析(ANOVA)。P < 0.05表示组间有显著性差异。
结果
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1 DFO@CuS纳米粒的理化性质
本研究根据以往的报道制备了中空硫化铜纳米粒, 利用其具有较大的比表面积和载药能力强等特点, 负载了促血管生成药物去铁胺(DFO)[19, 21]。CuS、DFO@CuS纳米粒的透射电镜结构如图 1A、B所示, 两者均具有致密的球壳结构、中部有明显的空洞, 证明成功合成了中空CuS纳米粒, 且载药过程不会影响其结构。图 1C为CuS纳米粒载药前后的粒径分布, CuS、DFO@CuS纳米粒的平均粒径分别为(180.5 ± 4.5) nm、(200.9 ± 8.6) nm, 与之前的报道一致。图 1D展现了CuS纳米粒载药前后的电位变化情况, 由图可以看出DFO、CuS和DFO@CuS纳米粒的平均Zeta电位分别为-8.413 ± 0.7681、-14.37 ± 2.301和-19.73 ± 1.795 mV, 提示DFO可能被包载于CuS纳米粒中。进一步地, 通过紫外分光光度计扫描发现(图 1E), DFO在226 nm左右具有最大吸收峰, 而DFO@CuS纳米粒在相同的位置处出现了特征吸收峰, 这证明DFO被成功包载到了其中。此外载药后DFO@CuS纳米粒在近红外区(800~1 100 nm) 仍具有较强的吸收峰, 说明负载DFO不会改变CuS纳米粒优异的光热转换性能。接着通过FeCl3比色法测量了上清中DFO的浓度, 并计算了包封率、载药量。结果显示, DFO@CuS纳米粒的包封率为24.93%、载药量为19.91%。
2 DFO@CuS纳米粒光热性能验证
目前对PTT的研究通常使用近红外一区(700~900 nm) 的激光作为辐射源。然而近些年来有研究发现, 近红外二区(1 000~1 700 nm) 在诊断与治疗方面更有优势, 因为它在穿透深度和光热转换效率方面优于近红外一区[30]。此外, 根据DFO@CuS纳米粒的紫外可见吸收光谱显示, 该纳米粒在800~1 100 nm有着较高的吸收峰, 因此本研究选用了980 nm激光器进行光热实验。如图 2A、B所示, DFO@CuS纳米粒与空白对照组相比具有良好的光热转换性能, 此外CuS纳米粒载药前后光热性能未发生显著变化。然后研究了在2.0 W·cm-2功率密度下不同浓度DFO@CuS纳米粒溶液的温度变化情况。结果显示(图 2C), 随着DFO@CuS纳米粒溶液浓度的增加, 溶液温度也相应上升。为了验证DFO@CuS纳米粒的光热稳定性, 进行了3个循环的“开-关”实验。结果如图 2D所示, 在3个“开-关”周期内DFO@CuS纳米粒的光热性能无显著变化, 光热稳定性良好。以上结果可以看出, DFO@CuS纳米粒可以作为理想的光热剂应用于抗菌治疗当中。
3 DFO@CuS纳米粒体外抗菌研究
为了验证DFO@CuS纳米粒的光热抗菌效果, 本研究使用了平板计数法进行评价。图 3A、C是耐甲氧西林金葡菌(MRSA) 和铜绿假单胞菌(PA) 在不同浓度DFO@CuS纳米粒经近红外光照射作用下的平板涂布图, 以不光照为对照组。由图可知, 在相同近红外光照射下(980 nm, 2.0 W·cm-2, 5 min), 随着DFO@CuS纳米粒浓度的不断增高, 培养基上的菌落逐渐减少, 在达到200 µg·mL-1浓度时, 平板上几乎没有菌落生成, 而不光照组的菌落数几乎没有发生改变, 这说明了该纳米粒本身不具有抗菌效果, 只有在近红外光照射下才具有良好的抗菌性能。此外本研究对平板上的菌落数进行了统计分析, 从图 3B、D可以看出, 在不光照组中, 纳米粒浓度的变化并不会导致菌落数发生明显改变。而在光照组中, 随着纳米粒浓度的提高。抗菌效果得到显著增强, 在DFO@CuS纳米粒浓度为100 µg·mL-1时, 抑菌率分别达到90.6%与84.4%。因此, 通过体外抗菌实验证明了该纳米粒具有优异的光热抗菌性能。
4 DFO@CuS纳米粒的细胞毒性
为了验证DFO@CuS纳米粒的生物相容性, 本研究使用CCK-8试剂盒评价了纳米粒对HUVECs的细胞毒性。由图 4A所示, 在与DFO@CuS纳米粒孵育24 h后, 当质量浓度小于200 µg·mL-1时, DFO@CuS纳米粒对HUVECs细胞并没有明显的杀伤效果; 而质量浓度大于200 µg·mL-1时, DFO@CuS纳米粒对HUVECs细胞的毒性显著增大, 最高浓度组(400 µg·mL-1) 的细胞存活率仅有63.4%。因此在后续的体外细胞实验中, 本研究将DFO@CuS纳米粒的浓度上限设定为100 µg·mL-1。
5 DFO@CuS纳米粒对HUVECs细胞迁移和血管生成能力的影响
通过细胞划痕实验来评价DFO@CuS纳米粒对HUVECs细胞迁移能力的影响。结果显示(图 4B), 在给予不同处理30 h后, 所有组的划痕面积均有不同程度的减小。然而与空白对照组细胞相比, 经DFO@CuS纳米粒处理后的细胞具有更窄的划痕面积, 表现出了更强的迁移能力。
血管生成是伤口愈合过程中的重要一环, 本研究用Matrigel基质胶模拟细胞外基质环境, 来评估HUVECs的成管能力。将与不同浓度的纳米粒处理24 h后的HUVECs细胞接种至Matrigel基质胶, 37 ℃孵育3 h后使用显微镜观察。结果如图 4C所示, 各组均出现了不同程度的血管形成特征。空白对照组仅出现了少量的管形结构, 大部分细胞仍分布分散。而经DFO@CuS纳米粒处理后的HUVECs细胞, 形成的管型更加致密, 并围成环状, 这是血管生成后期的典型特征, 说明血管化程度高。使用Image J血管生成插件对照片进行分析(图 4D), 发现新生血管的节点数和管长度都呈现出明显浓度依赖效应, 随着DFO@CuS纳米粒浓度的增高, 血管化程度也不断增高, 表明DFO@CuS纳米粒能够促进HUVECs细胞的小管形成, 证明其具有加速血管新生的能力。
讨论
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正常伤口愈合过程通常分为炎症期、增殖期与重塑期三个阶段, 而在糖尿病患者伤口愈合中, 由于长期的高糖环境导致粒细胞功能受损, 糖尿病伤口更易发生细菌感染, 细菌产生的毒力因子又会进一步加重炎症反应。此外, 由于晚期糖基化终产物的产生, 会损害内皮细胞的功能, 导致新生血管的减少, 不利于营养物质的运输与代谢产物的排出。这些因素导致糖尿病患者的伤口长期处在炎症阶段, 无法顺利进入增殖期与重塑期, 逐渐形成慢性伤口。因此, 有效抑制细菌生长与促进血管生成是加速糖尿病伤口愈合的关键。
本研究通过水热法制备了具有中空结构的CuS纳米粒, 并以此为载体包载了促血管生成药物DFO, 成功构建了一种新型的给药系统(DFO@CuS纳米粒)。该纳米粒具有良好的光热性能及较低的细胞毒性。通过体外抗菌实验表明, DFO@CuS纳米粒在近红外光照射下可以有效抑制多种细菌生长。通过体外细胞实验表明, DFO@CuS纳米粒可以促进HUVECs细胞的迁移与成管, 具有良好的促血管生成效应。因此, DFO@CuS纳米粒同时具有光热抗菌及促血管生成作用, 这种联合增效的治疗策略有望为慢性感染性糖尿病溃疡的治疗提供新方法。
在此基础上, 本课题组后续将进一步研究DFO@CuS纳米粒在糖尿病溃疡创面的应用, 鉴于水凝胶敷料具有保持伤口处湿润、方便给药等优势, 本课题组计划使用水凝胶对DFO@CuS纳米粒进行包载, 以便于该纳米粒在糖尿病溃疡治疗中的应用。此外, 由于DFO稳定性弱, CuS纳米粒与水凝胶的包载可能会提高DFO的稳定性, 而CuS纳米粒的光热效应可以将皮肤屏障打破, 有利于促进DFO的透皮吸收, 这些内容将是本课题组后续研究的方向。
作者贡献: 沈海俊和乔屹提出研究思路及设计研究方案; 乔屹、张纯和马燕娥负责进行实验; 陈嘉玲负责收集和分析数据; 乔屹负责撰写论文; 沈海俊负责最终版本修订。
利益冲突: 所有作者声明没有利益冲突。
基金
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  • 国家自然科学基金资助项目(82072044)
  • 江苏高校“青蓝工程”资助(2022年)
文献
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2023年第58卷第9期
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doi: 10.16438/j.0513-4870.2023-0062
  • 接收时间：2023-01-18
  • 首发时间：2025-11-21
  • 出版时间：2023-09-12
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  • 收稿日期：2023-01-18
  • 修回日期：2023-05-23
基金
国家自然科学基金资助项目(82072044)
江苏高校“青蓝工程”资助(2022年)
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    江苏大学医学院, 江苏 镇江 212013

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*沈海俊, E-mail:
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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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