Properties of gelatin-polyethylene glycol hydrogel loaded with silver nanoparticle Chlorella and its effects on healing of infected full-thickness skin defect wounds in mice
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摘要:
目的 探讨负载银纳米颗粒（AgNP）小球藻的明胶/聚乙二醇水凝胶（以下简称复合水凝胶）的性能及其对小鼠全层皮肤缺损感染创面愈合的作用。 方法 该研究为实验研究。制备单纯明胶/聚乙二醇水凝胶（以下简称单纯水凝胶）和复合水凝胶，大体观察2种水凝胶分别在55、37 ℃以及808 nm近红外光照射下的外观和可注射性；采用电子万能试验机测试2种水凝胶在室温下的拉应力-应变性能、压应力-应变性能以及复合水凝胶在80%最大压应力下的循环压应力-应变性能。分别于磷酸盐缓冲液（PBS）、单纯水凝胶和复合水凝胶中滴加金黄色葡萄球菌或大肠埃希菌菌液。取部分滴加了金黄色葡萄球菌或大肠埃希菌菌液的复合水凝胶，予近红外光照射5 min。将各样品孵育6 h后，采用稀释涂布平板法检测并计算2种细菌培养24 h的死亡率（样本数为5）。取海军军医大学第一附属医院泌尿外科收治的1名6岁健康男童包皮环切术后废弃包皮组织，采用酶解法提取分离原代人成纤维细胞（HFb），常规培养至第3~6代，用于后续细胞实验。制备终质量浓度分别为100.0、50.0、25.0、12.5、0 mg/mL的复合水凝胶浸提液，用其培养HFb，培养24 h后采用细胞计数试剂盒8检测细胞增殖活性，样本数为3。取20只6~8周龄雌性C57BL/6J小鼠，在每只鼠背部制作1个全层皮肤缺损创面，在创面上涂抹金黄色葡萄球菌菌液进行感染。将感染小鼠按照随机数字表法分为空白对照组、单纯水凝胶组、复合水凝胶组、联合处理组，每组5只小鼠，分别采用PBS、单纯水凝胶、复合水凝胶、复合水凝胶+光照（用波长808 nm近红外光照射5 min）处理其创面。于伤后0（首次处理创面后即刻）、3、7、14 d，大体观察各组小鼠创面渗出及愈合情况并计算伤后7、14 d的创面愈合率，样本数为5。伤后14 d，行苏木精-伊红染色观察小鼠创面组织病理学变化。 结果 单纯水凝胶和复合水凝胶在55 ℃时均为溶液状态，降温到37 ℃后均呈凝胶态；近红外光照射2种水凝胶后，仅复合水凝胶升温，可再次回到溶液状态，即具有可注射性。复合水凝胶的最大拉应力可达301.42 kPa，对应的应变为87.19%；最大压应力可达413.79 kPa，对应的应变为91.67%，均与单纯水凝胶的拉伸和压缩性能相近。复合水凝胶经过10次压缩循环后，其最大压应力仍可达第1次压应力的84.1%。培养24 h，金黄色葡萄球菌经单纯水凝胶处理后的死亡率明显高于经PBS处理后（ P<0.05）；大肠埃希菌和金黄色葡萄球菌仅经复合水凝胶处理后的死亡率均明显高于经单纯水凝胶处理后（ P<0.05）；大肠埃希菌和金黄色葡萄球菌经复合水凝胶+光照处理后的死亡率均明显高于仅经复合水凝胶处理后（ P<0.05）。培养24 h，与用终质量浓度0 mg/mL复合水凝胶浸提液培养相比，用终质量浓度25.0、50.0 mg/mL复合水凝胶浸提液培养的HFb的增殖活力均明显增强（ P<0.05），用终质量浓度100.0 mg/mL复合水凝胶浸提液培养的HFb的增殖活力明显减弱（ P<0.05）。伤后0、3 d，空白对照组、单纯水凝胶组小鼠创面中的脓性分泌物均较多，复合水凝胶组小鼠创面中仅有少量渗出液，而联合处理组小鼠创面中未见明显感染。伤后7、14 d，单纯水凝胶组小鼠创面愈合率均明显高于空白对照组（ P<0.05），复合水凝胶组小鼠创面愈合率均明显高于单纯水凝胶组（ P<0.05），联合处理组小鼠创面愈合率均明显高于复合水凝胶组（ P<0.05）。伤后14 d，空白对照组小鼠创面有较多的炎症细胞浸润、未见新生的上皮层；单纯水凝胶组小鼠创面中新生上皮长度短，且存在少量炎症细胞；复合水凝胶组小鼠创面中有连续的新生上皮形成，以及大量未成熟的肉芽组织；联合处理组小鼠创面有连续的新生上皮形成，未成熟肉芽组织较少。 结论 制备的复合水凝胶具有良好的温敏性、光热性和可注射性，以及良好的机械性能、抗菌性能和生物相容性，可促进小鼠全层皮肤缺损感染创面愈合。 Abstract:Objective To explore the properties of gelatin-polyethylene glycol hydrogel loaded with silver nanoparticle (AgNP) Chlorella (hereinafter referred to as the composite hydrogel) and its effects on healing of infected full-thickness skin defect wounds in mice. Methods The research was an experimental research. The simple gelatin-polyethylene glycol hydrogel (hereinafter referred to as the simple hydrogel) and the composite hydrogel were prepared, and the appearance and injectability of the two hydrogels were observed at 55 and 37 ℃, and under the irradiation of 808 nm near-infrared light, respectively. An electronic universal testing machine was employed to assess the tensile and compressive stress-strain properties of both types of hydrogels at room temperature. Additionally, the cyclic compressive stress-strain properties of the composite hydrogel were examined at 80% of the maximum compressive stress. Staphylococcus aureus or Escherichia coli solution was added to phosphate buffer solution (PBS), simple hydrogel, and composite hydrogel, respectively. The part of composite hydrogel containing Staphylococcus aureus or Escherichia coli solution was irradiated with near-infrared light for 5 minutes. After each sample was incubated for 6 h, the dilution plating method was used to detect and calculate the mortality rates of the two bacteria at 24 h of culture ( n=5). The discarded foreskin tissue was taken from a 6-year-old healthy boy admitted to the Department of Urology of the First Affiliated Hospital of Naval Medical University for circumcision. Primary human fibroblasts (HFbs) were isolated using the enzyme extraction method, routinely cultured to the 3 rd to 6 th passages for subsequent cellular experiments. Composite hydrogel extracts with final mass concentrations of 100.0, 50.0, 25.0, 12.5, and 0 mg/mL were respectively prepared and used to culture HFbs, and the cell proliferation after 24 h of culture was detected using a cell counting kit 8 ( n=3). A total of twenty 6-8 weeks old C57BL/6J female mice were utilized, and a full-thickness skin defect was surgically created on the back of each mouse. The wounds were infected with Staphylococcus aureus solution. The infected mice were divided into blank control group, simple hydrogel group, composite hydrogel group, and combined treatment group according to the random number table, and the wounds were treated with PBS, simple hydrogel, composite hydrogel, and composite hydrogel+light irradiation (under the irradiation of 808 nm near-infrared light for 5 min), respectively, with 5 mice in each group. On post injury day (PID) 0 (immediately after the first wound treatment), 3, 7, and 14, an overall assessment of wound exudation and healing were conducted, and the wound healing rates on PID 7 and 14 were calculated ( n=5). On PID 14, hematoxylin-eosin staining was performed to observe histopathological changes in the mouse wound. Results Both simple hydrogel and composite hydrogel were in a solution state at 55 ℃ and transition to a gel state when cooling to 37 ℃. After the two hydrogels were irradiated by near-infrared light, only the composite hydrogel reheated up and returned to the solution state again with injectability. The maximum tensile stress of the composite hydrogel was up to 301.42 kPa, with a corresponding strain of 87.19%; the maximum compressive stress was up to 413.79 kPa, with a corresponding strain of 91.67%, which was similar to the tensile and compressive properties of the simple hydrogel. After 10 compression cycles, the maximum compressive stress of the composite hydrogel still reached 84.1% of the first compressive stress. After 24 h of culture, the mortality rate of Staphylococcus aureus treated with simple hydrogel was significantly higher than that treated with PBS ( P<0.05); the mortality rates of Escherichia coli and Staphylococcus aureus treated with composite hydrogel alone were significantly higher than those treated with simple hydrogel ( P<0.05); the mortality rates of Escherichia coli and Staphylococcus aureus treated with composite hydrogel+light irradiation were significantly higher than those treated with composite hydrogel alone ( P<0.05). After 24 h of culture, compared with that cultured in composite hydrogel immersion solution with final mass concentration of 0 mg/mL, the proliferation activity of HFbs cultured in composite hydrogel immersion solution with final mass concentrations of 25.0 and 50.0 mg/mL was significantly enhanced ( P<0.05), while the proliferation activity of HFbs cultured in composite hydrogel immersion solution with final mass concentration of 100 mg/mL was significantly decreased ( P<0.05). On PID 0 and 3, more purulent secretions were seen in the wounds of mice in blank control group and simple hydrogel group, while only a small amount of exudate was observed in the wounds of mice in composite hydrogel group, and no obvious infection was observed in the wounds of mice in combined treatment group. On PID 7 and 14, the wound healing rates of mice in simple hydrogel group were significantly higher than those in blank control group ( P<0.05); the wound healing rates of mice in composite hydrogel group were significantly higher than those in simple hydrogel group ( P<0.05); the wound healing rates in combined treatment group were significantly higher than those in composite hydrogel group ( P<0.05). On PID 14, the wounds of mice in blank control group exhibited a high infiltration of inflammatory cells with no new epithelial layer observed; the wounds of mice in simple hydrogel group displayed a short length of newly formed epithelium with a small amount of inflammatory cells; the wounds of mice in composite hydrogel group exhibited continuous formation of new epithelium and a large amount of immature granulation tissue; the wounds of mice in combined treatment group showed continuous epithelialization with less immature granulation tissue. Conclusions The prepared composite hydrogel exhibits excellent thermosensitivity, photothermal properties, and injectability, as well as excellent mechanical properties, antibacterial properties, and biocompatibility, and can promote the healing of infected full-thickness skin defect wounds in mice. -
Key words:
- Hydrogel /
- Nanoparticles /
- Bacterial infections /
- Photothermal therapy /
- Green synthesis /
- Silver nano /
- Wound repair
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参考文献
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1 单纯小球藻和银纳米颗粒（AgNP）小球藻的微观形貌及AgNP小球藻含有的化学元素 扫描电子显微镜×10 000。1A.单纯小球藻颗粒均匀；1B.AgNP小球藻颗粒均匀，且颗粒表面可见明显小突起；1C.AgNP小球藻的元素分布图，左上指示银元素、右上指示氧元素、左下指示磷元素、右下指示碳元素
2 2种水凝胶随温度及近红外光光照的变化。2A.55 ℃时单纯水凝胶（左）和复合水凝胶（右）均为溶液状态；2B.37 ℃时单纯水凝胶（左）和复合水凝胶（右）均为凝胶态；2C.凝胶态的复合水凝胶经光照4 min后恢复溶液状态，具有可注射性
注：单纯水凝胶指明胶/聚乙二醇水凝胶，复合水凝胶指负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶
3 采用电子万能试验机检测2种水凝胶的机械性能。3A.2种水凝胶的拉应力-应变曲线；3B.2种水凝胶的压应力-应变曲线；3C.复合水凝胶的循环压应力-应变曲线
注：单纯水凝胶指明胶/聚乙二醇水凝胶，复合水凝胶指负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶
4 稀释涂布平板法检测2种细菌在4种处理方式下培养24 h的生长情况。4A、4B、4C、4D.分别为大肠埃希菌在磷酸盐缓冲液、单纯水凝胶、复合水凝胶、复合水凝胶+波长808 nm近红外光照射5 min处理下的生长情况；4E、4F、4G、4H.分别为金黄色葡萄球菌在前述4种处理方式下的生长情况，图4E、4F、4G、4H的菌落数变化情况分别与图4A、4B、4C、4D相近
注：单纯水凝胶指明胶/聚乙二醇水凝胶，复合水凝胶指负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶
5 用不同终质量浓度的复合水凝胶浸提液培养24 h后人成纤维细胞增殖活力比较（样本数为3，
注：复合水凝胶指负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶；总体比较，F=44.48，P<0.001；与0 mg/mL相比，aP<0.05
6 4组小鼠伤后各时间点全层皮肤缺损感染创面愈合情况。6A、6B、6C、6D.分别为空白对照组在伤后0、3、7、14 d创面愈合情况；6F、6G、6H、6I.分别为单纯水凝胶组创面在前述时间点的愈合情况；6K、6L、6M、6N.分别为复合水凝胶组创面在前述时间点的愈合情况；6P、6Q、6R、6S.分别为联合处理组创面在前述时间点的情况，图6E、6J、6O、6T中绿色边界和蓝色边界内的面积均基本呈缩小趋势
注：空白对照组、单纯水凝胶组、复合水凝胶组和联合处理组分别采用磷酸盐缓冲液、明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶+波长808 nm近红外光照射5 min处理；图中圆形参照物直径为1 cm；图6E、6J、6O、6T为ImageJ软件处理后的量化图，图中红色、黄色、绿色、蓝色边界内面积分别表示伤后0（首次处理创面后即刻）、3、7、14 d的创面面积
7 伤后14 d的4组小鼠全层皮肤缺损感染创面组织病理学情况 苏木精-伊红×5。7A、7B、7C、7D.分别为空白对照组、单纯水凝胶组、复合水凝胶组、联合处理组创面，与图7A、7B、7C相比，图7D的新生上皮连续且未成熟肉芽组织少
注：空白对照组、单纯水凝胶组、复合水凝胶组和联合处理组分别采用磷酸盐缓冲液、明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶+波长808 nm近红外光照射5 min处理
表1 稀释涂布平板法检测2种细菌在4种处理方式下培养24 h的死亡率（%，
表1. The mortality rates of the two bacteria cultured under the four treatments for 24 h detected by the dilution plating method
处理方式 样本数 大肠埃希菌 金黄色葡萄球菌 磷酸盐缓冲液 5 0±7.2 0±3.1 单纯水凝胶 5 10.6±2.4 19.6±5.2 c 复合水凝胶 5 62.8±8.3 a 46.6±6.4 a 复合水凝胶+光照 5 94.2±4.0 b 90.6±5.7 b F值 221.80 224.80 P值 <0.001 <0.001 注：单纯水凝胶指明胶/聚乙二醇水凝胶，复合水凝胶指负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶，光照指采用波长808 nm近红外光照射5 min；与单纯水凝胶相比， a P<0.05；与复合水凝胶相比， b P<0.05；与磷酸盐缓冲液相比， c P<0.05 
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表2 伤后7、14 d的4组小鼠全层皮肤缺损感染创面愈合率比较（%，
表2. Comparison of healing rates of infected full-thickness skin defect wounds of mice in 4 groups on post injury day 7 and 14
组别 样本数 7 d 14 d 空白对照组 5 31.2±3.5 56.6±7.0 单纯水凝胶组 5 43.8±5.8 72.0±5.2 复合水凝胶组 5 58.6±3.8 81.8±5.3 联合处理组 5 71.4±3.8 94.2±2.6 F值 81.56 43.09 P值 <0.001 <0.001 P 1值 0.001 <0.001 P 2值 <0.001 0.015 P 3值 0.001 0.002 注：空白对照组、单纯水凝胶组、复合水凝胶组和联合处理组分别采用磷酸盐缓冲液、明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶、负载银纳米颗粒小球藻的明胶/聚乙二醇水凝胶+波长808 nm近红外光照射5 min处理；时间因素主效应， F=1 471.00， P<0.001；处理因素主效应， F=64.15， P<0.001；二者交互作用， F=3.91， P=0.028； P 1值为单纯水凝胶组与空白对照组比较所得； P 2值为复合水凝胶组与单纯水凝胶组比较所得； P 3值为联合处理组与复合水凝胶组比较所得
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