黑色素纳米颗粒-丝素蛋白-单宁酸水凝胶的制备及其抗氧化性能检测

Preparation of melanin nanoparticles-silk fibroin-tannic acid hydrogel and detection of its anti-oxidant properties

  • 摘要:
    背景 在牙周软组织再生过程中,由于各种原因引起的氧化应激状态常导致组织修复迟滞,而传统修复材料对此疗效不佳。制备具有抗氧化功能的水凝胶材料,有希望为牙周软组织生物材料修复提供更多的选择。
    目的 制备黑色素纳米颗粒(melanin nanoparticles,MNPs) - 单宁酸(tannic acid,TA) - 丝素蛋白(silk fibroin,SF)复合水凝胶,检测MNPs含量对水凝胶微观形貌、抗氧化性能和细胞相容性的影响。
    方法 制备MNPs,将不同浓度的MNPs加入到SF,加入TA化学交联,得到MNPs-SF-TA复合水凝胶。根据水凝胶中MNPs浓度分为实验组MNPs1-SF-TA(1 mg/mL)、MNPs2-SF-TA(2 mg/mL)、MNPs4-SF-TA(4 mg/mL)和对照组SF-TA(0 mg/mL)。通过扫描电镜(scanning electron microscope,SEM)观察水凝胶表面形貌;1,1-二苯基-2-三硝基苯肼(2,2-Diphenyl-1-picrylhydrazyl,DPPH)法、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid),ABTS法和Cu2+法检测其自由基清除能力和抗氧化能力;采用CCK-8和Calcein-AM/PI活/死细胞荧光染色法检测该水凝胶材料的细胞相容性。
    结果 成功制备了 MNPs和MNPs-SF-TA复合水凝胶。DPPH、ABTS及Cu2+法均显示MNPs-SF-TA水凝胶对自由基的清除能力和抗氧化能力随MNPs浓度的升高而提高(P<0.05),4 mg/mL时达峰值。CCK-8结果显示:随观察时间延长,吸光度增强,细胞增殖活性增强;相同时间下各组细胞增殖活力随MNPs浓度升高而降低,MNPs2-SF-TA组显著高于MNPs4-SF-TA组(P<0.05);Calcein-AM/PI荧光染色结果与此一致。
    结论 成功制备了MNPs-SF-TA复合水凝胶,其中MNPs浓度为2 mg/mL时,MNPs-SF-TA水凝胶具有较强的活性氧清除能力和良好的细胞相容性,有望成为牙周软组织修复的新型生物替代材料。

     

    Abstract:
    Background During the process of periodontal soft tissue regeneration, oxidative stress caused by various factors often leads to delayed tissue repair. Traditional restorative materials have shown limited efficacy in addressing this issue. Therefore, the development of hydrogel materials with antioxidant properties holds promising potential to offer more choices for the repair of periodontal soft tissue biomaterials.
    Objective To prepare a composite hydrogel of melanin nanoparticles-silk fibroin-tannic acid (MNPs-SF-TA) and assess the impact of melanin nanoparticles (MNPs) content on the hydrogel's microstructure, antioxidant performance, and cellular compatibility.
    Methods MNPs were prepared and different concentrations of MNPs were added to silk fibroin (SF), followed by the addition of tannic acid (TA) for chemical crosslinking, resulting in the formation of MNPs-SF-TA composite hydrogel. The hydrogel was divided into experimental groups: MNPs1-SF-TA (1 mg/mL), MNPs2-SF-TA (2 mg/mL), MNPs4-SF-TA (4 mg/mL), and a control group: SF-TA (0 mg/mL). The surface morphology of the hydrogel was observed using scanning electron microscopy (SEM). The radical scavenging ability and antioxidant capacity of the hydrogel were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay, and Cu2+ assay. The cellular compatibility of the hydrogel material was assessed using the CCK-8 assay and Calcein-AM/PI live/dead cell fluorescent staining method.
    Results MNPs and MNPs-SF-TA composite hydrogel were successfully prepared. The DPPH, ABTS, and Cu2+ assays all demonstrated that the scavenging ability of free radicals and antioxidant capacity of MNPs-SF-TA hydrogel increased with the increasing concentration of MNPs (P < 0.05), reaching a peak at 4 mg/mL. The CCK-8 results showed that cell proliferation activity, as indicated by the increased absorbance, enhanced with prolonged observation time. With the same observation time, the cell proliferation activity of each group decreased with the increasing concentration of MNPs, and the MNPs2-SF-TA group was significantly higher than the MNPs4-SF-TA group (P < 0.05). The results of Calcein-AM/PI fluorescence staining were consistent with these findings.
    Conclusion MNPs-SF-TA composite hydrogel is successfully prepared, and the hydrogel exhibits strong ROS scavenging ability and good cellular compatibility when the concentration of MNPs is 2 mg/mL. Therefore, MNPs-SF-TA hydrogel holds great promise as a novel biomaterial for periodontal soft tissue repair and may serve as a potential alternative to traditional materials in this field.

     

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