| 苗春杰,史鑫扬,高文月,等.海藻酸钠基酶促纳米反应器的制备及其催化活性评价[J].中国海洋药物,2025,44(6):53-62. |
| 海藻酸钠基酶促纳米反应器的制备及其催化活性评价 |
| Preparation and Catalytic Activity Evaluation of Sodium Alginate-based Enzyme-Promoted Nanoreactors |
| 投稿时间：2024-04-18 修订日期：2024-05-20 |
| DOI：10.13400/j.cnki.cjmd.2025.06.010 |
| 中文关键词: 生物大分子药物 酶 纳米水凝胶 海藻酸钠 壳聚糖 |
| English Keywords:biomacromolecular drugs enzymes hydrogel sodium alginate chitosan |
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| 中文摘要: |
| 目的 构建基于海藻酸钠（Sodium alginate，SA）纳米水凝胶（Nanogels，NGs）的纳米反应器系统，旨在有效负载葡萄糖氧化酶（Glucose oxidase，GOx）和辣根过氧化物酶（Horseradish peroxidase，HRP），同时确保酶的活性与稳定性，为实现其在抗肿瘤、炎症治疗等应用的安全性与高效性奠定研究基础。方法 采用乳化-溶剂蒸发-离子交联法和直接离子交联法制备未负载酶的海藻酸钠纳米水凝胶（SA NGs）和负载单一酶（GOx或HRP）的海藻酸钠纳米水凝胶（GOx@SA NGs和HRP@SA NGs）。通过动态光散射技术测定其粒径和Zeta电位，评估制备条件对SA NGs性质的影响。采用Amplex Red分析法对GOx@SA NGs和HRP@SA NGs进行活性评估，测定其米氏常数（Km）、最大反应速度（Vmax）、转换数（Kcat）和专一性常数（Kcat/Km）。结果 乳化-溶剂蒸发-离子交联法制备的SA NGs表现出较小的粒径（89.6 nm ± 1.5 nm）和接近中性的Zeta电位（3.1 mV ± 1.3 mV）。通过直接离子交联法制备的SA NGs，其粒径可通过调节SA的浓度从450.4 nm ± 33.4 nm降至320.6 nm ± 2.2 nm，Zeta电位均维持较大的负电位（-34.3 mV ± 1.5 mV ~ -32.7 6 mV ± 2.3 mV）。通过离子交联法制备的GOx@SA NGs和HRP@SA NGs显示出在负载酶后粒径有所增加，分别为403.4 nm ± 1.1 nm和359.9 nm ± 10.2 nm。尽管GOx@SA NGs和HRP@SA NGs的平均Zeta电位略有降低，分别为-23.0 mV ± 0.7 mV和-24.7 mV ± 4.7 mV，但仍维持在-20 mV以上，表明负载酶的SA NGs在溶液中保持相对良好的稳定性。酶活性测试结果显示，两种酶在SA NGs中均保持高活性，且能有效催化目标反应。结论 本研究成功制备了具有优良稳定性的海藻酸钠纳米水凝胶，并证明这些纳米水凝胶可以作为生物酶的高效载体，负载的GOx和HRP保持了良好的活性，此纳米反应器可用于多种生物酶的负载及其催化应用。 |
| English Summary: |
| Objective To construct a nanoreactor system based on sodium alginate nanogels (SA NGs), aimed at effectively loading glucose oxidase (GOx) and horseradish peroxidase (HRP) while ensuring the enzymes" activity and stability. This lays a research foundation for the safety and efficiency of their applications in anti-tumor and inflammation therapy. Methods Emulsion-solvent evaporation-ion crosslinking method and direct ion crosslinking method were used to prepare unloaded SA NGs and enzyme-loaded SA NGs (GOx@SA NGs and HRP@SA NGs). The size and Zeta potential of the NGs were measured by dynamic light scattering to evaluate the impact of preparation conditions on SA NGs characteristics. The activity of GOx@SA NGs and HRP@SA NGs was assessed using the Amplex Red assay, determining their Michaelis constant (Km), maximum reaction velocity (Vmax), turnover number (Kcat), and specificity constant (Kcat/Km). Results SA NGs prepared by emulsion-solvent evaporation-ion crosslinking method showed smaller particle size (89.6 nm ± 1.5 nm) and near-neutral Zeta potential (3.1 mV ± 1.3 mV). Direct ion crosslinking method-produced SA NGs had their particle size reduced from 450.4 nm ± 33.4 nm to 320.6 nm ± 2.2 nm by adjusting the concentration of SA, maintaining a relatively high negative Zeta potential (-34.3 mV ± 1.5 mV to -32.6 mV ± 2.3 mV). Enzyme loading resulted in increased particle size for GOx@SA NGs and HRP@SA NGs, at 403.4 nm ± 1.1 nm and 359.9 nm ± 10.2 nm respectively. Although the Zeta potential of GOx@SA NGs and HRP@SA NGs slightly decreased to -23.0 mV ± 0.7 mV and -24.7 mV ± 4.7 mV, respectively, they still maintained above -20 mV, indicating relatively good stability of enzyme-loaded SA NGs in solution. Enzyme activity tests showed that both enzymes retained high activity within SA NGs and could effectively catalyze target reactions. Conclusion This study successfully synthesized sodium alginate nanogels with excellent stability and demonstrated that these nanogels can act as efficient carriers for bioenzymes. The encapsulated GOx and HRP retained good activity, making this nanoreactor suitable for the loading and catalytic applications of various bioenzymes. |
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