摘要
背景:MXene纳米颗粒具备出色的亲水性、生物相容性和抗菌特性,被广泛应用于创面、肿瘤、神经修复和心血管等治疗领域,目前尚不清楚MXene纳米颗粒对糖尿病创面愈合的作用。目的:考察MXene纳米颗粒Ti_(3)C_(2)T_(x)的体外抗氧化、抗炎和光热抗菌活性,以及对糖尿病小鼠创面的修复效果。方法:①体外实验:采用MTT法检测不同质量浓度Ti_(3)C_(2)T_(x)对小鼠成纤维细胞(NIH-3T3)的毒性作用。将NIH-3T3细胞暴露在H_(2)O_(2)中,采用MTT法检测不同质量浓度Ti_(3)C_(2)T_(x)对NIH-3T3细胞的保护作用。将NIH-3T3细胞暴露在H_(2)O_(2)中,分析光照(或不光照)处理下Ti_(3)C_(2)T_(x)(20μg/mL)对NIH-3T3细胞活性氧生成的影响。将RAW264.7巨噬细胞分3组处理:对照组、脂多糖组与脂多糖+Ti_(3)C_(2)T_(x)组,采用实时定量PCR法检测细胞中特定基因(CD86、白细胞介素6、CD206、精氨酸酶1)的表达。将大肠杆菌(或金黄色葡萄球菌)分3组处理:对照组、Ti_(3)C_(2)T_(x)组、Ti_(3)C_(2)T_(x)光照组,利用平板菌落计数法计算细菌存活率。②体内实验:通过腹腔注射链脲佐菌素的方法构建ICR小鼠糖尿病模型,造模成功后在小鼠背部建立全层皮肤缺损创面,随机分3组干预:对照组(n=6)、Ti_(3)C_(2)T_(x)组(n=6)和Ti_(3)C_(2)T_(x)光照组(n=6),观察创面愈合情况,干预后第7天进行创面组织CD31、CD206免疫组化染色,干预后第7,14天进行创面组织苏木精-伊红与Masson染色。将Ti_(3)C_(2)T_(x)溶液注射至ICR小鼠皮下,在光照(或非光照)后,通过血生化检测分析Ti_(3)C_(2)T_(x)对小鼠的毒性作用。结果与结论:①体外实验:Ti_(3)C_(2)T_(x)质量浓度在5-160μg/mL范围内对NIH-3T3细胞无毒性作用,在质量浓度20μg/mL时可增加NIH-3T3细胞存活率。10-80μg/mL的Ti_(3)C_(2)T_(x)可显著提升H_(2)O_(2)干预下的NIH-3T3细胞存活率。Ti_(3)C_(2)T_(x)可显著抑制H_(2)O_(2)干预下NIH-3T3细胞活性氧的生成,光照处理可进一步提升Ti_(3)C_(2)T_(x)抑制活性氧生成的作用。Ti_(3)C_(2)T_(x)能够有效抑制由脂多糖诱导的巨噬细胞炎症,促进细胞向具有抗炎特性的M2型巨噬细胞转化。Ti_(3)C_(2)T_(x)与Ti_(3)C_(2)T_(x)光照均可显著抑制大肠杆菌和金黄色葡萄球菌的生长,并且Ti_(3)C_(2)T_(x)光照的抑制效果更显著。②体内实验:创面大体与组织学分析显示,Ti_(3)C_(2)T_(x)与Ti_(3)C_(2)T_(x)光照均可促进糖尿病小鼠创面的愈合,并且Ti_(3)C_(2)T_(x)光照的促进作用更显著。免疫组化染色结果显示,Ti_(3)C_(2)T_(x)与Ti_(3)C_(2)T_(x)光照均可抑制糖尿病创面的炎症反应并促进血管的生成,并且Ti_(3)C_(2)T_(x)光照的作用更显著。血生化检测结果显示,Ti_(3)C_(2)T_(x)与光照对小鼠无明显毒性作用。③结果表明,Ti_(3)C_(2)T_(x)借助独特的光热效应能够高效地展现抗氧化、抗炎和抗菌特性,促进糖尿病小鼠创面的愈合。
BACKGROUND:MXene nanoparticles,due to their unique hydrophilicity,biocompatibility,and antibacterial properties,are widely used in wound,tumor,nerve repair,and cardiovascular treatments.However,it is still unclear what effect MXene nanoparticles have on diabetic wound healing.OBJECTIVE:To investigate the in vitro antioxidant,anti-inflammatory and photothermal antibacterial properties of MXene nanoparticles Ti_(3)C_(2)T_(x) as well as their effect on wound repair in diabetic mice.METHODS:(1)In vitro experiments:The cytotoxicity of Ti_(3)C_(2)T_(x) nanoparticles on mouse fibroblasts(NIH-3T3)at various concentrations was evaluated using the methyl thiazolyl tetrazolium(MTT)assay.NIH-3T3 cells were exposed to H_(2)O_(2),and the MTT assay was used to detect the protective effects of different mass concentrations of Ti_(3)C_(2)T_(x) on NIH-3T3 cells.NIH-3T3 cells were exposed to H_(2)O_(2),and the effect of Ti_(3)C_(2)T_(x)(20μg/mL)on the generation of reactive oxygen species in NIH-3T3 cells was analyzed under illumination(or no illumination)treatment.RAW264.7 macrophages were divided into three groups:control group,lipopolysaccharide group,and lipopolysaccharide+Ti_(3)C_(2)T_(x) group.Real-time quantitative PCR was used to detect the expression of specific genes(CD86,interleukin 6,CD206,arginase 1)in the cells.Escherichia coli(or Staphylococcus aureus)were divided into three groups:control group,Ti_(3)C_(2)T_(x) group,and Ti_(3)C_(2)T_(x) illumination group.The bacterial survival rate was calculated by plate colony counting method.(2)In vivo experiments:Streptozotocin was administered intraperitoneally to ICR mice to induce a diabetic condition.After successful modeling,a full-thickness skin defect wound was created on the back of the mice using a circular punch.The experiment was divided into three groups:control group(n=6),Ti_(3)C_(2)T_(x) group(n=6),and Ti_(3)C_(2)T_(x) illumination group(n=6).The wound healing was observed,and CD31 and CD206 immunohistochemical staining of wound tissue was performed on day 7 after intervention.Hematoxylin-eosin staining and Masson staining of wound tissue were performed on days 7 and 14 after intervention.Ti_(3)C_(2)T_(x) solution was injected subcutaneously into ICR mice.After illumination(or non-illumination)exposure,the toxic effects of Ti_(3)C_(2)T_(x) on mice were analyzed by blood biochemical detection.RESULTS AND CONCLUSION:(1)In vitro experiments:Ti_(3)C_(2)T_(x) showed no cytotoxicity on NIH-3T3 cells at mass concentrations ranging from 5-160μg/mL.It increased the survival rate of NIH-3T3 cells at a mass concentration of 20μg/mL.Ti_(3)C_(2)T_(x) at 10-80μg/mL significantly improved the survival rate of NIH-3T3 cells under H_(2)O_(2) intervention.Ti_(3)C_(2)T_(x) significantly inhibited the generation of reactive oxygen species in NIH-3T3 cells under the intervention of H_(2)O_(2),and illumination treatment further enhanced the effect of Ti_(3)C_(2)T_(x) on inhibiting the generation of reactive oxygen species.Ti_(3)C_(2)T_(x) effectively inhibited macrophage inflammation induced by lipopolysaccharide and promoted the transformation of cells into M2 macrophages with anti-inflammatory properties.Both Ti_(3)C_(2)T_(x) and Ti_(3)C_(2)T_(x) illumination significantly inhibited the growth of Escherichia coli and Staphylococcus aureus,and the inhibitory effect of Ti_(3)C_(2)T_(x) illumination was more significant.(2)In vivo experiments:Gross and histological analyses of the wound surface showed that both Ti_(3)C_(2)T_(x) and Ti_(3)C_(2)T_(x) illumination promoted wound healing in diabetic mice,and the promotion effect of Ti_(3)C_(2)T_(x) irradiation was more significant.Immunohistochemical staining results showed that both Ti_(3)C_(2)T_(x) and Ti_(3)C_(2)T_(x) illumination inhibited the inflammatory response in diabetic wounds and promoted angiogenesis,and the effect of Ti_(3)C_(2)T_(x) illumination was more significant.Blood biochemical test results showed that Ti_(3)C_(2)T_(x) and illumination had no obvious toxic effects on mice.(3)These results indicate that Ti_(3)C_(2)T_(x) nanoparticles efficiently promote the healing of skin wounds in a diabetic mouse model through antioxidation,anti-inflammation,and antibacterial actions via photothermal effects.
作者
李美运
刘森
陈开元
施灵
宋美陈
曹家洪
武艳
于晶
Li Meiyun;Liu Sen;Chen Kaiyuan;Shi Ling;Song Meichen;Cao Jiahong;Wu Yan;Yu Jing(College of Life Sciences,Mudanjiang Medical University,Mudanjiang 157001,Heilongjiang Province,China;Department of Clinical Pathology,General Hospital of Southern Theater Command of People’s Liberation Army of China,Guangzhou 510030,Guangdong Province,China;Department of Endocrinology,Hongqi Hospital,Mudanjiang Medical College,Mudanjiang 157001,Heilongjiang Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第28期6052-6060,共9页
Chinese Journal of Tissue Engineering Research
基金
牡丹江市指导性科技计划项目(HT2022JG125),项目负责人:武艳
黑龙江省省属高等学校基本科研业务费科研项目(2021-KYYWF-0515),项目负责人:于晶。
