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

doi:10.13481/j.1671-587X.20240206

摘要/Abstract

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）%。结论 MSC-Exo@AgNP/HA-ADH/ODex水凝胶具有优异的抗菌性能，能够有效促进人表皮HaCaT细胞的增殖，可作为一种安全有效的生物敷料，用于皮肤创伤的治疗。

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

Abstract:

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 100% when treated with the dilution ratios of 0，1， 2， 4， 8， 16， 32， and 64 of culture medium-extract of HA-ADH/ODex hydrogel. For the AgNP/HA-ADH/ODex hydrogel extracted with dilution ratio of 8 and above， the survival rates were greater than 85%. The spread plate method results showed that the antibe terial rates of AgNP/HA-ADH/ODex and MSC-Exo@AgNP/HA-ADH/ODex hydrogels against Staphylococcus aureus （S. aureus） and Escherichia coli （E. coli）， and the antibacterial rates against S.aureus and E. coli were （96.44±0.16）%，（96.62±0.16）% and （95.73±0.28）%，（95.58±0.14）%， respectively. The CCK-8 assay results showed that the MSC-Exo@AgNP/HA-ADH/ODex hydrogel promoted the proliferation of the HaCaT cells，and the proliferation rate was （115.00 ± 7.42）%. Conclusion The MSC-Exo@AgNP/HA-ADH/ODex hydrogel exhibits the excellent antibacterial properties and good ability to promote the proliferation of human epidermal HaCaT cells； it is a safe and effective bio-dressing for the treatment of skin wounds.

Key words: Hyaluronic acid, Dextran, Silver nanoparticles, Mesenchymal stem cell exosome, Skin wound, Drug delivery

中图分类号:

R751

刘韬,傅家财,钟秤明,滕旭,齐玲. 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[J]. 吉林大学学报(医学版), 2024, 50(2): 336-345.

Tao LIU,Jiacai FU,Chengming ZHONG,Xu TENG,Ling QI. Construction of exosome and AgNPs-loaded antibacterial hydrogel and its effect on proliferation of human epidermal HaCaT cells[J]. Journal of Jilin University(Medicine Edition), 2024, 50(2): 336-345.

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