【摘要】 目的 构建负载磷脂酶A2（PLA2）抑制剂和铜掺杂碳量子点（Cu-CDs）的复合微针系统，并评价其对氮芥化合物2-氯乙基乙基硫醚（CEES）致伤和细菌感染的复合皮肤创面的治疗作用。方法 （1）微针制备与表征：将PLA2抑制剂伐瑞拉迪与Cu-CDs共载于透明质酸基质中，经模板法制备复合微针。采用光学显微镜、扫描电子显微镜观察微针形态；通过小鼠背部皮肤刺入实验评估微针的溶解性能和生物安全性；利用透射电子显微镜及Zeta电位分析仪对Cu-CDs进行表征。（2）体外抗菌实验：将Cu-CDs 0.1、1、10、50、100和200 mg·L^-1分别与大肠杆菌（EC）或金黄色葡萄球菌（SA）共培养，通过平板计数法评估其抗菌效果。（3）体外抗炎与抗毒实验：采用CCK-8法筛选CEES 1.5、3、6、12和24 mmol·L^-1对人永生化角质形成细胞（HaCaT）的损伤浓度，以此作为细胞损伤模型浓度，并考察伐瑞拉迪62.5、125、250和500μmol·L^-1干预后的细胞存活率；采用ELISA检测炎症因子白细胞介素6（IL-6）、IL-1β和肿瘤坏死因子α（TNF-α）的水平。（4）体内药效学评价：小鼠随机分为染菌组（EC或SA）、染毒组(CEES 150μL·kg^-1，背部覆膜法染毒)、染菌染毒组（CEES-EC或CEES-SA，染菌24 h后染毒）、染毒给药组（CEES-T，染毒后用微针处理创面）、染菌染毒给药组（CEES-EC-T或CEES-SA-T，染菌染毒后用微针处理创面）。分别于染菌第3、5、7、9和11天记录各组的创面面积；于第7和12天检测其创面血流量；于第12天通过革兰氏染色观察抗菌情况，并采用HE染色和免疫荧光染色进行病理评价；于第3、5、7和9天记录CEES-SA和CEES-SA-T组创面面积，并于第7天检测其创面血流量。结果 （1）成功制备形貌均一的方锥形复合微针，针高约1.2 mm，具有良好的溶解性与生物安全性。（2） Cu-CDs 100 mg·L^-1对2种测试菌均呈现抑菌效果。（3） CEES 3 mmol·L^-1时HaCaT细胞存活率降至60%左右，而伐瑞拉迪500μmol·L^-1干预可使细胞存活率恢复至对照水平，并显著降低炎症因子IL-6和IL-1β水平。（4）在EC与SA感染的皮肤复合模型中，载药微针治疗组的创面愈合速度均显著快于相应染菌染毒组。第12天，CEES-T组和CEES-EC-T组创面血流量明显高于CEES组、EC组和CEES-EC组。第7和9天，CEES-T组和CEES-EC-T组创面面积较CEES-EC组均显著减小；第11天，CEES-EC-T组创面面积与CEES-EC组相比显著减小。革兰氏染色结果显示，CEES-EC组皮肤创伤部位可见大量红色阳性区域，CEES-EC-T组无明显红色。与CEES、EC和CEES-EC组相比，CEES-T和CEES-EC-T组皮肤组织中炎症因子IL-6表达明显降低，Ⅰ型胶原蛋白含量升高。与CEES-SA组相比，第7和9天CEES-SA-T组创面面积显著减小，创面血流量明显增加。结论 本研究成功构建了共载PLA2抑制剂与Cu-CDs的复合微针系统，该微针通过协同抗炎与抗菌作用，显著促进CEES损伤和细菌感染的复合皮肤创面愈合。更多还原

【Abstract】 OBJECTIVE To construct a composite microneedle system that co-encapsulates a phospholipase A2（PLA2） inhibitor and copper-doped carbon dots（Cu-CDs）, and to investigate its therapeutic efficacy against complex skin wounds induced by the nitrogen mustard analog 2-chloroethyl ethyl sulfide（CEES） superimposed with bacterial infection. METHODS（1） Fabrication and characterization: Varespladib, a selective PLA2 inhibitor, and Cu-CDs were simultaneously incorporated into a hyaluronic acid matrix via a template-mediated process to produce composite microneedles, the morphology of which was examined under optical and scanning electron microscopy while skin penetration capacity,dissolution behavior, and biosafety were evaluated using a mouse dorsal skin model. Cu-CDs were characterized via transmission electron microscopy and zeta potential measurement.（2） In vitro antibacterial evaluation: Escherichia coli（EC） or Staphylococci aureus（SA） was co-incubated with Cu-CDs at graded concentrations 0.1, 1, 10, 50, 100 and 200 mg·L^-1, and the surviving bacterial colonies were enumerated using the plate counting method.（3） In vitro anti-inflammatory and cytoprotective assays: A cytotoxicity profile of CEES was established in immortalized human keratinocytes（HaCaT） across a concentration range of 1.5, 3, 6, 12 and 24 mmol·L^-1using the CCK-8 assay to define the injury model.Following intoxication, cells were treated with varespladib at 62.5, 125, 250 and 500 μmol·L^-1before cell viability was measured. Levels of pro-inflammatory cytokines, including interleukin-6（IL-6）, IL-1β,and tumor necrosis factor-α（TNF-α）, were quantified by ELISA.（4） In vivo pharmacodynamic assessment: Mice were randomly divided into bacterial infection groups（EC or SA）, an intoxication group(CEES applied dorsally under occlusive film at 150 μL · kg^-1), combined infection-intoxication groups（CEES-EC or CEES-SA, with intoxication initiated 24 h post-inoculation）, an intoxication-treatment group（CEES-T, receiving microneedle intervention after intoxication）, and combined injury-treatment groups（CEES-EC-T or CEES-SA-T, treated after the combined insult）. Wound closure was quantified on days 3, 5, 7, 9 and 11 post-infection in CEES, EC, CEES-EC, CEES-T, and CEES-EC-T groups,and regional blood perfusion was measured on days 7 and 12. On day 12, wound tissue specimens were harvested for Gram staining to assess bacterial burden, hematoxylin-eosin staining was used for histological examination, and immunofluorescence staining was adopted to determine IL-6 expression and collagen deposition. For the CEES-SA and CEES-SA-T cohorts, wound area dynamics were recorded on days 3, 5, 7 and 9, with perfusion evaluated on day 7. RESULTS（1） Uniform, square-pyramidal composite microneedles with an approximate height of 1.2 mm were produced that were characterized by robust skin penetrability, rapid dissolution and excellent biosafety.（2） Cu-CDs at 100 mg·L^-1exerted potent antibacterial activity against both bacterial strains.（3） Exposure to 3 mmol · L^-1CEES reduced HaCaT cell viability to roughly 60%, whereas subsequent administration of 500 μmol·L-1 varespladib restored cellular viability and markedly suppressed IL-6 secretion.（4） In both EC-and SA-infected complex wound models, wound contraction was significantly accelerated in microneedle-treated groups compared with their untreated combined injury counterparts. On day 12, blood perfusion in the CEES-T and CEES-EC-T groups was significantly elevated compared to the CEES, EC, and CEES-EC groups.Wound areas in the CEES-T and CEES-EC-T groups became significantly smaller on days 7 and 9 compared with the CEES-EC group, and this reduction persisted in the CEES-EC-T group on day 11.Gram staining revealed extensive red-positive signals indicative of bacterial colonization in CEES-EC wounds, which was virtually absent in the CEES-EC-T group. Compared with the CEES, EC, and CEES-EC groups, pronounced downregulation of IL-6 and augmented deposition of typeⅠcollagen in the regenerating dermis were observed in CEES-T and CEES-EC-T groups. Furthermore, compared with the CEES-SA group, CEES-SA-T treatment resulted in significantly smaller wound areas on days7 and 9, accompanied by a marked increase in wound blood flow intensity. CONCLUSION A composite microneedle system integrating a PLA2 inhibitor with Cu-CDs has been constructed. By synergizing anti-inflammatory and antibacterial mechanisms, this transdermal platform can substantially accelerate the healing of complex cutaneous wounds arising from CEES intoxication compounded by bacterial infection.更多还原