Effects of reactive oxygen species-responsive antibacterial microneedles on the full-thickness skin defect wounds with bacterial colonization in diabetic mice
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摘要:
目的 研究活性氧响应性抗菌微针对糖尿病小鼠细菌定植全层皮肤缺损创面的影响。 方法 采用实验研究方法。合成活性氧响应性交联剂N1-（4-溴苄基）-N3-（4-溴苯基）-N1，N1，N3，N3-四甲基丙烷-1，3-二胺（TSPBA），混合相应成分制成聚乙烯醇-TSPBA（PVA-TSPBA）微针、PVA-ε-聚赖氨酸（ε-PL）-TSPBA微针、PVA-TSPBA-透明质酸钠（SH）微针、PVA-ε-PL-TSPBA-SH微针。将PVA-TSPBA微针分别置于单纯磷酸盐缓冲液（PBS）和含过氧化氢的PBS中，观察浸泡0（即刻）、3、7、10 d微针降解情况，表示其活性氧响应性。将用含过氧化氢的LB培养基培养的金黄色葡萄球菌标准菌株与大肠埃希菌标准菌株各自按随机数字表法（分组方法下同）分为空白对照组（不行任何处理，下同）以及与含相应浓度ε-PL的PVA-ε-PL-TSPBA微针共培养的0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组，培养24 h，观察细菌生长情况并计算细菌相对存活率（样本数为3）。将对数生长期的小鼠成纤维细胞系3T3细胞（生长周期下同）分为空白对照组以及用含相应浓度ε-PL的PVA-ε-PL-TSPBA微针浸提液培养的0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组，培养24 h，用光学显微镜观察细胞生长情况，用细胞计数试剂盒8（CCK-8）法检测并计算细胞相对存活率（以此表示细胞毒性，样本数为6）。取PVA-TSPBA微针与PVA-TSPBA-SH微针，用光学显微镜观察2种微针形貌，用微机控制电子万能试验机检测2种微针机械性能（以临界力表示，样本数为6）。取6只6~8周龄雄性BALB/c小鼠（性别、鼠龄下同），分为PVA-TSPBA组与PVA-TSPBA-SH组（每组3只），用相应微针垂直按压背部皮肤1 min后，观察按压完成后0、10、20 min皮肤情况。另取3T3细胞，分为空白对照组，用含相应浓度ε-PL的PVA-ε-PL-TSPBA微针浸提液培养的0 g/L ε-PL组、单纯5.0 g/L ε-PL组，用含5.0 g/L ε-PL的PVA-ε-PL-TSPBA-SH微针浸提液培养的5.0 g/L ε-PL+SH组，CCK-8法检测并计算培养24、48、72 h细胞相对存活率，以此表示细胞增殖活性（样本数为6）。取18只BALB/c小鼠，通过高糖高脂饮食联合链脲佐菌素注射诱导为糖尿病小鼠模型后，分为无菌敷贴组、0 g/L ε-PL+SH组与5.0 g/L ε-PL+SH组（每组6只），在每只小鼠背部制作全层皮肤缺损创面后滴加金黄色葡萄球菌溶液，制成糖尿病小鼠细菌定植全层皮肤缺损创面模型，0 g/L ε-PL+SH组、5.0 g/L ε-PL+SH组小鼠创面覆盖含相应浓度ε-PL的PVA-ε-PL-TSPBA-SH微针后，3组小鼠创面均外覆无菌手术敷贴。于伤后0、3、7、12 d观察创面愈合情况，计算伤后3、7、12 d创面愈合率；伤后12 d，取创面及创缘皮肤组织行苏木精-伊红染色，观察新生上皮生长及炎症细胞浸润情况。对数据行单因素方差分析、重复测量方差分析、Mann-Whitney U检验、Bonferroni法。 结果 随着浸泡时间的延长，置于含过氧化氢PBS中的PVA-TSPBA微针逐渐溶解并于浸泡10 d完全降解，置于单纯PBS中的PVA-TSPBA微针仅发生溶胀而未溶解。培养24 h，5.0 g/L ε-PL组、10.0 g/L ε-PL组金黄色葡萄球菌未见生长，10.0 g/L ε-PL组大肠埃希菌未见生长；1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组金黄色葡萄球菌相对存活率较空白对照组明显降低（P<0.05或P<0.01），5.0 g/L ε-PL组、10.0 g/L ε-PL组大肠埃希菌相对存活率较空白对照组明显降低（P<0.01）。培养24 h，空白对照组、0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组细胞生长状态良好，组间细胞相对存活率相近（P>0.05）。PVA-TSPBA微针与PVA-TSPBA-SH微针的针体均呈四棱锥形，排列整齐，其中PVA-TSPBA-SH微针的针体更立体、棱角更分明。PVA-TSPBA-SH微针的临界力明显高于PVA-TSPBA微针（Z=3.317，P<0.01）。PVA-TSPBA-SH组小鼠按压完成后0 min微针穿透皮肤，10 min后针孔部分消失，20 min后针孔完全消失；PVA-TSPBA组微针未能穿透小鼠皮肤。培养24、48、72 h，5.0 g/L ε-PL+SH组细胞增殖活性均明显高于空白对照组（P<0.05或P<0.01）。无菌敷贴组小鼠创面愈合速度缓慢，渗出较多；0 g/L ε-PL+SH组小鼠创面愈合速度前期与无菌敷贴组相近，后期较无菌敷贴组加快，渗出中等；5.0 g/L ε-PL+SH组小鼠创面愈合较另2组快，渗出不多。5.0 g/L ε-PL+SH组小鼠伤后3、7、12 d创面愈合率分别为（40.6±4.2）%、（64.3±4.1）%、（95.8±2.4）%，明显高于无菌敷贴组的（20.4±2.7）%、（38.9±2.2）%、（59.1±6.2）%与0 g/L ε-PL+SH组的（21.6±2.6）%、（44.0±1.7）%、（82.2±5.3）%（P<0.01）；0 g/L ε-PL+SH组小鼠伤后7、12 d创面愈合率明显高于无菌敷贴组（P<0.05或P<0.01）。伤后12 d，5.0 g/L ε-PL+SH组小鼠创面几乎完全上皮化且炎症细胞浸润较少，0 g/L ε-PL+SH组小鼠创面部分上皮化且伴大量炎症细胞浸润，无菌敷贴组小鼠创面未见明显上皮化且伴大量炎症细胞浸润。 结论 基于TSPBA、聚乙烯醇、ε-PL及SH制备的复合微针可顺利刺穿小鼠皮肤并能通过缓慢响应创面中的活性氧从而溶解释放抗菌物质，抑制创面细菌定植，促进糖尿病小鼠细菌定植全层皮肤缺损创面修复。 Abstract:Objective To study the effects of reactive oxygen species (ROS)-responsive antibacterial microneedles (MNs) on the full-thickness skin defect wounds with bacterial colonization in diabetic mice. Methods Experimental research methods were adopted. The ROS-responsive crosslinker N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) was first synthesized, and then the polyvinyl alcohol (PVA)-TSPBA MNs, PVA-ε-polylysine (ε-PL)-TSPBA MNs, PVA-TSPBA-sodium hyaluronate (SH) MNs, and PVA-ε-PL-TSPBA-SH MNs were prepared by mixing corresponding ingredients, respectively. The PVA-TSPBA MNs were placed in pure phosphate buffer solution (PBS) and PBS containing hydrogen peroxide, respectively. The degradation of MNs immersed for 0 (immediately), 3, 7, and 10 days was observed to indicate their ROS responsiveness. The standard strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) cultured in Luria-Bertani medium containing hydrogen peroxide were divided according to the random number table (the same grouping method below) into blank control group (without any treatment, the same below) and 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group with which PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL were co-cultured, respectively. Bacterial growth was observed after 24 h of culture, and the relative survival rate of bacteria was calculated (n=3). The mouse fibroblast cell line 3T3 cells at logarithmic growth stage (the same growth stage below) were divided into blank control group and 0 g/L ε-PL group, 1.0 g /L ε-PL group, 5.0 g /L ε-PL group, and 10.0 g /L ε-PL group in which cells were cultured in medium with the extract from PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, respectively. Cell growth was observed after 24 h of culture by optical microscopy, and the relative survival rate of cells was detected and calculated by cell counting kit 8 (CCK-8) assay to indicate the cytotoxicity (n=6). Both PVA-TSPBA MNs and PVA-TSPBA-SH MNs were taken, the morphology of the two kinds of MNs was observed by optical microscopy, and the mechanical properties of the two kinds of MNs were tested by microcomputer controlled electronic universal testing machine (denoted as critical force, n=6). Six male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into PVA-TSPBA group and PVA-TSPBA-SH group, with 3 mice in each group. After pressing the skin on the back of mice vertically with the corresponding MNs for 1 minute, the skin condition was observed at 0, 10, and 20 min after pressing. Another batch of 3T3 cells were divided into blank control group, 0 g/L ε-PL group and simple 5.0 g/L ε-PL group which were cultured with the extract of PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, and 5.0 g/L ε-PL+SH group which were cultured with the extract of PVA-ε-PL-TSPBA-SH MNs with 5.0 g/L ε-PL. The CCK-8 assay was performed to detect and calculate the relative survival rate of cells cultured for 24, 48, and 72 h to indicate the cell proliferation activity (n=6). Eighteen BALB/c mice were induced into diabetic mice model by high-sugar and high-fat diet combined with streptozotocin injection and then divided into sterile dressing group, 0 g/L ε-PL+SH group, and 5.0 g/L ε-PL+SH group, with 6 mice in each group. A full-thickness skin defect wound was made on the back of each mouse, and S. aureus solution was added to make a full-thickness skin defect wound with bacterial colonization model for diabetic mouse. The wounds of mice in 0 g/L ε-PL+SH group and 5.0 g/L ε-PL+SH group were covered with PVA-ε-PL-TSPBA-SH MNs with the corresponding concentration of ε-PL, and the wounds of mice in the 3 groups were all covered with sterile surgical dressings. The wound healing was observed on post injury day (PID) 0, 3, 7, and 12, and the wound healing rate on PID 3, 7, and 12 was calculated. On PID 12, the skin tissue of the wound and the wound margin were stained with hematoxylin and eosin to observe the growth of new epithelium and the infiltration of inflammatory cells. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Mann-Whitney U test, and Bonferroni test. Results With the extension of the immersion time, the PVA-TSPBA MNs in PBS containing hydrogen peroxide gradually dissolved and completely degraded after 10 days of immersion. The PVA-TSPBA MNs in pure PBS only swelled but did not dissolve. After 24 h of culture, there was no growth of S. aureus in 5.0 g/L ε-PL group or 10.0 g/L ε-PL group, and there was no growth of E. coli in 10.0 g/L ε-PL group. The relative survival rate of S. aureus was significantly lower in 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group than in blank control group (P<0.05 or P<0.01). The relative survival rate ofE. coli was significantly lower in 5.0 g/L ε-PL group and 10.0 g/L ε-PL group than in blank control group (P<0.01). After 24 h of culture, the cells in blank control group, 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group all grew well, and the relative survival rate of cells was similar among the groups (P>0.05). The needle bodies of PVA-TSPBA MNs and PVA-TSPBA-SH MNs were both quadrangular pyramid-shaped and neatly arranged, and the needle bodies of PVA-TSPBA-SH MNs was more three-dimensional and more angular. The critical force of PVA-TSPBA-SH MNs was significantly higher than that of PVA-TSPBA MNs (Z=3.317, P<0.01). The MNs in PVA-TSPBA+SH group penetrated the skin of mice at 0 min after pressing, and the pinholes partially disappeared after 10 min and completely disappeared after 20 min, while the MNs in PVA-TSPBA group failed to penetrate the skin of mice. After 24, 48, and 72 h of culture, the proliferation activity of the cells in 5.0 g/L ε-PL+SH group was significantly higher than that of blank control group (P<0.05 or P<0.01). In sterile dressing group, the wounds of mice healed slowly and exuded more. The wound healing speed of mice in 0 g/L ε-PL+SH group was similar to that of sterile dressing group in the early stage but was faster than that of sterile dressing group in the later stage, with moderate exudation. The wound healing of mice in 5.0 g/L ε-PL+SH group was faster than that in the other two groups, with less exudation. The wound healing rates of mice in 5.0 g/L ε-PL+SH group were (40.6±4.2)%, (64.3±4.1)%, and (95.8±2.4)% on PID 3, 7, and 12, which were significantly higher than (20.4±2.7)%, (38.9±2.2)%, and (59.1±6.2)% in sterile dressing group and (21.6±2.6)%, (44.0±1.7)%, and (82.2±5.3)% in 0 g/L ε-PL+SH group (P<0.01). The wound healing rates of mice in 0 g/L ε-PL+SH group on PID 7 and 12 were significantly higher than those in sterile dressing group (P<0.05 or P<0.01). On PID 12, the wounds of mice in 5.0 g/L ε-PL+SH group were almost completely epithelialized with less inflammatory cell infiltration, the wounds of mice in 0 g/L ε-PL+SH group were partially epithelialized with a large number of inflammatory cell infiltration, and no obvious epithelialization but a large number of inflammatory cell infiltration was found in the wounds of mice in sterile dressing group. Conclusions The composite MNs prepared by TSPBA, PVA, ε-PL, and SH can successfully penetrate mouse skin and slowly respond to ROS in the wound to resolve and release antibacterial substances, inhibit bacterial colonization, and promote the repair of full-thickness skin defect wounds with bacterial colonization in diabetic mice. -
1 聚乙烯醇-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺微针在2种磷酸盐缓冲液（PBS）中的溶解情况 图中标尺为1 mm。1A、1B、1C、1D.分别为于单纯PBS中浸泡0、3、7、10 d微针形态，浸泡0 d时微针沉入底部，浸泡3、7、10 d微针逐渐溶胀但未溶解；1E、1F、1G、1H.分别为于含终物质的量浓度0.25 mmol/L过氧化氢的PBS中浸泡0、3、7、10 d微针形态，浸泡0 d时与图1A相近，浸泡3、7 d逐渐溶解，浸泡10 d完全溶解
2 5组金黄色葡萄球菌、大肠埃希菌培养24 h在LB琼脂培养板上的生长情况 图中标尺为1 cm。2A、2B、2C、2D、2E.分别为空白对照组、0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组金黄色葡萄球菌生长情况，图2A中菌落较多，图2B中菌落数与图2A相近，图2C中菌落数少于图2A，图2D、2E未见细菌生长；2F、2G、2H、2I、2J.分别为空白对照组、0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组大肠埃希菌生长情况，图2F中菌落较多，图2G中菌落数与图2F相近，图2H中菌落数略少于图2F，图2I中菌落数明显少于图2F，图2J未见细菌生长
注：各ε-聚赖氨酸（ε-PL）组细菌与含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺微针共培养
3 小鼠成纤维细胞系3T3细胞培养24 h的生长情况 光学显微镜×200，图中标尺为100 μm。3A、3B、3C、3D、3E.分别为空白对照组、0 g/L ε-PL组、1.0 g/L ε-PL组、5.0 g/L ε-PL组、10.0 g/L ε-PL组，各组细胞均正常增殖，细胞生长状态良好，细胞排列有序，形态均一
注：各ε-聚赖氨酸（ε-PL）组细胞用含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺微针浸提液培养
4 2种活性氧响应性微针形态学观察。4A.聚乙烯醇-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺（PVA-TSPBA）微针排列整齐，针体为四棱锥形，但呈空心状 图中标尺为400 µm；4B.PVA-TSPBA微针针尖不够锐利，针体呈空心状 光学显微镜×200，图中标尺为100 µm；4C.PVA-TSPBA-透明质酸钠（SH）微针排列整齐，针体为四棱锥形且针体饱满 图中标尺为400 µm；4D.PVA-TSPBA-SH微针针尖锐利，针体饱满 光学显微镜×200，图中标尺为100 µm
5 2组小鼠皮肤上按压相应活性氧响应性微针1 min后各时间点皮肤情况 图中标尺为1 mm。5A、5B、5C.分别为聚乙烯醇-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺（PVA-TSPBA）-透明质酸钠组按压完成后0（即刻）、10、20 min，0 min针孔清晰可见，10 min时部分皮肤已恢复正常但部分皮肤仍留有针孔，20 min时针孔完全消失；5D.PVA-TSPBA组按压完成后0 min，未见明显针孔
6 3组糖尿病小鼠细菌定植全层皮肤缺损创面伤后各时间点愈合情况 图中标尺为2 mm。6A、6B、6C、6D.分别为无菌敷贴组伤后0、3、7、12 d创面情况，愈合速度缓慢，伤后12 d仍有大部分创面未愈合；6E、6F、6G、6H.分别为0 g/L ε-PL+SH组伤后0、3、7、12 d创面情况，前1周内愈合速度较缓慢，图6E除表面覆盖微针外与图6A基本一致，图6F创面面积与图6B相近，图6G创面面积与图6C相近，图6H创面面积较图6D缩小；6I、6J、6K、6L.分别为5.0 g/L ε-PL+SH组伤后0、3、7、12 d创面情况，愈合较快，图6I与图6E基本一致，图6J创面面积较图6B、6F明显缩小，图6K创面面积较图6C与图6G明显缩小，图6L创面已基本愈合
注：0 g/L ε-聚赖氨酸（ε-PL）+透明质酸钠（SH）组、5.0 g/L ε-PL+SH组创面覆盖含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺-SH微针
7 3组糖尿病小鼠细菌定植全层皮肤缺损创面伤后12 d组织学观察 苏木精-伊红×40，图中标尺为50 μm。7A.无菌敷贴组创面未见明显上皮化，有大量炎症细胞（蓝紫色）浸润；7B.0 g/L ε-PL+SH组创面部分上皮化，有大量炎症细胞浸润；7C.5.0 g/L ε-PL+SH组创面几乎完全上皮化且炎症细胞浸润较少
注：黑色双向箭头示未上皮化区域；0 g/L ε-聚赖氨酸（ε-PL）+透明质酸钠（SH）组、5.0 g/L ε-PL+SH组创面覆盖含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺-SH微针
表1 4组小鼠成纤维细胞系3T3细胞培养各时间点相对存活率比较（%，
组别 样本数 24 h 48 h 72 h 空白对照组 6 100±11 130±17 204±14 0 g/L ε-PL组 6 134±17 135±15 216±14 单纯5.0 g/L ε-PL组 6 138±16 128±18 218±8 5.0 g/L ε-PL+SH组 6 140±14a 155±14b 239±12b 注：0 g/L ε-聚赖氨酸（ε-PL）组、单纯5.0 g/L ε-PL组分别用含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺（PVA-ε-PL-TSPBA）微针浸提液培养，最后一组用含5.0 g/L ε-PL的PVA-ε-PL-TSPBA-透明质酸钠（SH）微针浸提液培养；处理因素主效应，F=183.357，P<0.001；时间因素主效应，F=235.311，P<0.001；两者交互作用，F=0.593，P=0.702；与空白对照组比较，aP<0.01，bP<0.05 
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表2 3组糖尿病小鼠细菌定植全层皮肤缺损创面伤后各时间点愈合率比较（%，
组别 鼠数（只） 伤后3 d 伤后7 d 伤后12 d 无菌敷贴组 6 20.4±2.7 38.9±2.2 59.1±6.2 0 g/L ε-PL+SH组 6 21.6±2.6 44.0±1.7c 82.2±5.3a 5.0 g/L ε-PL+SH组 6 40.6±4.2ab 64.3±4.1ab 95.8±2.4ab 注：0 g/L ε-聚赖氨酸（ε-PL）+透明质酸钠（SH）组、5.0 g/L ε-PL+SH组创面覆盖含相应浓度ε-PL的聚乙烯醇-ε-PL-N1-(4-溴苄基)-N3-(4-溴苯基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺-SH微针；处理因素主效应，F=711.051，P<0.001；时间因素主效应，F=717.622，P<0.001；两者交互作用，F=13.481，P<0.001；与无菌敷贴组比较，aP<0.01，cP<0.05；与0 g/L ε-PL+SH组比较，bP<0.01
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