载外泌体银纳米颗粒抗菌水凝胶的构建及其对人表皮HaCaT细胞增殖的影响  

Construction of exosome and AgNPs-loaded antibacterial hydrogel and its effect on proliferation of human epidermal HaCaT cells

作　　者：刘韬 傅家财 钟秤明 滕旭 齐玲 LIU Tao;FU Jiacai;ZHONG Chengming;TENG Xu;QI Ling(College of Pharmcy,Dali University,Dali 671000,China;ffiliated Qingyuan Hospital,Quangzhou Medical University,Institute of Digestive Disease,People’s Hospital,Qingyuan City,Qingyuan 511518,China;School of Basic Medical Sciences,Gannan Medical University,Ganzhou 341000,China)

机构地区：[1]大理大学药学院,云南大理671000 [2]广州医科大学附属清远医院清远市人民医院,广东清远511500 [3]赣南医学院基础医学院,江西赣州341000

出　　处：《吉林大学学报（医学版）》2024年第2期336-345,共10页Journal of Jilin University：Medicine Edition

基　　金：广东省卫健委医学科学技术研究基金项目(A2023208);广东省清远市人民医院医学科研基金项目(20220329,20190220)。

摘　　要：目的:构建负载间充质干细胞外泌体(MSC-Exo)和银纳米颗粒(AgNPs)抗菌水凝胶,初步探讨该水凝胶的抗菌活性及促人表皮HaCaT细胞增殖的效果,阐明其在皮肤损伤治疗方面的应用潜力。方法:利用氧化葡聚糖(ODex)和改性透明质酸(HA-ADH)制备基础水凝胶骨架,以十六烷基三甲基溴化铵(CTAB)为稳定剂制备AgNPs;培养间充质干细胞(MSC)获取MSC-Exo。构建AgNP/HA-ADH/ODex水凝胶和MSC-Exo@AgNP/HA-ADH/ODex水凝胶。通过BCA法评估外泌体的负载率,选用动态光散射(DLS)分析、Zeta电位分析、紫外-可见分光光度计表征AgNPs形貌、表面带电情况和结构,利用红外光谱表征HA-ADH/ODex水凝胶骨架的结构组成,通过透射电子显微镜(TEM)、纳米流式检测仪和蛋白免疫印迹(Western blotting)法对MSC-Exo进行鉴定,CCK-8法检测HA-ADH/ODex水凝胶和AgNP/HA-ADH/ODex水凝胶的细胞存活率及MSC-Exo@AgNP/HA-ADH/ODex水凝胶对HaCaT细胞的细胞增殖率的影响,平板涂布法和比浊法评估AgNP/HA-ADH/ODex水凝胶的抗菌活性。结果:DLS分析、Zeta电位分析和紫外分光光度计检测,成功制备了粒径大小均一、表面带正电的AgNPs。红外光谱检测,HA-ADH/ODex水凝胶制备成功。TEM、纳米流式检测和Western blotting法验证MSC-Exo成功提取,BCA法证实在MSC-Exo@AgNP/HA-ADH/ODex水凝胶中MSC-Exo负载率为77.8%。CCK-8法检测,稀释倍数为0、1、2、4、8、16、32和64的HA-ADH/ODex水凝胶浸提液处理的HaCaT细胞存活率接近100%。稀释8倍及以上的AgNP/HA-ADH/ODex水凝胶浸提液处理的HaCaT细胞存活率均大于85%。平板涂布法检测,AgNP/HA-ADH/ODex和MSC-Exo@AgNP/HA-ADH/ODex水凝胶具有明显的抗菌活性,其对金黄色葡萄球菌(S.aureus)的抗菌率分别为(96.44±0.16)%和(96.62±0.16)%,对大肠杆菌(E.coli)的抗菌率分别为(95.73±0.28)%和(95.58±0.14)%。CCK-8法检测,MSC-Exo@AgNP/HA-ADH/ODex水凝胶能够促进HaCaT细胞增殖,细胞增殖率为(115.00±7.42)%。结�Objective:To construct the antibacterial hydrogel containing mesenchymal stem cell exosomes(MSC-Exo)and silver nanoparticles(AgNPs),and to preliminarily clarify its antibacterial activity and proliferative effect on the human epidermal HaCaT cells,and to clarify its application potential in treatment of skin wound.Methods:The basic hydrogel scaffold was fabricated using oxidized dextran(ODex)and modified hyaluronic acid(HA-ADH).The AgNPs were synthesized with cetyltrimethylammonium bromide(CTAB)as the stabilizer,while the MSC-Exo was isolated from the cultured mesenchymal stem cells(MSC).The AgNP/HA-ADH/ODex hydrogel and MSC-Exo@AgNP/HA-ADH/ODex hydrogel were constructed.The loading rate of the Msc-Exo was evaluated through BCA assay;dynamic light scattering(DLS),Zeta potential measurement,and ultraviolet-visible spectroscope were used to analyze the morphology,the charged status of the AgNPs;fourier transform infrared spectra was used to detect the compositions of the HA-ADH/ODex hydrogel framework;transmission electron microscope(TEM),nanoparticle tracking analysis,and Western blotting methods were used to identify the presence of MSC-Exo;the cell survival rates of the HA-ADH/ODex hydrogel and the AgNP/HA-ADH/ODex hydrogel,and the effect of MSC-Exo@AgNP/HA-ADH/ODex hydrogel on the proliferation rate of HaCaT cells were detected by CCK-8 assay;the antibacterial efficacy of the AgNP/HA-ADH/ODex hydrogel was detected by spread plate method and turbidity measurement.Results:The DLS,Zeta potential,and ultraviolet-visible spectrum results showed the successful synthesis of uniform and positively charged AgNPs.The fourier transform infrared spectrometry results showed the HA-ADH/ODex hydrogel was properly formulated.The identification of MSC-Exo was verified by TEM,nanoparticle tracking,and Western blotting methods.The BCA assay results showed that the loading rate of the MSC-Exo on the MSC-Exo@AgNP/HA-ADH/ODex hydrogel was 77.8%.The CCK-8 assay results showed that the survival rates of the HaCaT cells were close to 1

关 键 词：透明质酸 葡聚糖 银纳米颗粒 间充质干细胞外泌体 皮肤创伤 药物递送 

分 类 号：R751[医药卫生—皮肤病学与性病学]