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 ther-apeutic efficacy against complex skin wounds induced by the nitrogen mustard analog 2-chloroethyl ethyl sulfide(CEES)superimposed with bacterial infection.METHODS ① Fabrication and character-ization: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 antibac-terial 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-1 using 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-1 before 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 assess-ment: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-pyra-midal composite microneedles with an approximate height of 1.2 mm were produced that were charac-terized by robust skin penetrability,rapid dissolution and excellent biosafety.② Cu-CDs at 100 mg·L-1 exerted potent antibacterial activity against both bacterial strains.③ Exposure to 3 mmol·L-1 CEES reduced HaCaT cell viability to roughly 60%,whereas subsequent administration of 500 μmol·L-1 vare-spladib 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 days 7 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.关键词
铜掺杂碳量子点/2-氯乙基乙基硫醚/磷脂酶A2抑制剂/伐瑞拉迪/创面/血流/复合微针Key words
copper-doped carbon dots/2-chloroethyl ethyl sulfide/phospholipase A2 inhibitor/varespladib/wound/blood flow/composite microneedle分类
医药卫生
