缓释型纳米抑藻颗粒在蓝藻细胞内外介质中的转运释放及毒性机制

It is a hotspot and difficulty in the field of environmental research to control algal blooms effectively and to control eutrophication water. The control of algae growth and reproduction by plant allelopathy with ecological security is concerned at home and abroad. In view of the fact that there are a lot of high ecological risk and low efficiency of algae inhibition in the process of algae control, this project aims to increase the binding amount of cyanobacterial cells to low dose of f allelochemicals as the breakthrough point. Referring to the design concept of nano slow release material, artemisinin, linoleic acid and gallic acid were used as targets of allelochemicals, and biodegradable nanoparticles of chitosan, alginate and cyclodextrin with the wide application were choosed as carrier to optimize and prepare algal inhibitory nanoparticles with high encapsulation efficiency and sustained release controlled release efficiency. The swelling, dispersion, drug release and degradation behavior of nanoparticles were studied to identify the key factors controlling nanoparticle release and dispersion and to elucidate the release mechanism and transport pathway of nanoparticles and allelochemicals in cyanobacteria cells. The combined effects of nano particles and allelochemicals on aggregation damage, physiological growth effect, antioxidant enzyme system and cytological mechanism of cyanobacteria cells were investigated to reveal the toxic mechanism of the synergistic induction effect of allelochemicals and nanoparticles on the death of cyanobacteria cells. The research results can provide new ideas for the control of cyanobacteria bloom.

蓝藻暴发性生长引发的灾害使有效控制水华、治理富营养化水体成为环境领域的研究热点和难点，生态安全的植物化感作用控制藻类生长繁殖受到国内外关注。针对化感物质在抑藻过程中存在高剂量生态风险大、低剂量抑藻效能差的问题，本项目以增加蓝藻细胞对低剂量化感物质的结合量为切入点，借鉴纳米缓释材料设计理念，以青蒿素、亚油酸和没食子酸作为目标抑藻化感物质，以应用广可降解纳米材料壳聚糖、海藻酸盐和环糊精作为缓释载体，优化制备具有高包埋率和缓释控释效能的抑藻纳米颗粒，研究纳米颗粒溶胀、分散团聚、药物释放及降解行为，识别调控纳米颗粒缓释和分散性能的关键因素，阐明纳米颗粒和化感物质在蓝藻细胞内外介质中的释放机理和转运途径，探究纳米颗粒与化感物质对蓝藻细胞团聚损伤、生理生长效应、抗氧化酶系统及细胞学机制的联合作用和持续作用，揭示化感物质、纳米颗粒协同诱导蓝藻细胞死亡的毒性机制，研究成果可为蓝藻水华控制提供新思路。

藻华暴发具有范围广、频次高、生物量高等特征，造成水生系统结构和功能严重失衡。同时，铜绿微囊藻作为优势产毒菌种，其生物量增加将增大藻毒素释放风险，进一步威胁人类健康。本项目以壳聚糖/海藻酸钠为包埋材料， 采用微胶囊技术制备改进青蒿素、亚油酸、茶多酚缓释微粒，从铜绿微囊藻胞质水平、生化分子水平、细胞形态结构等方面，阐释纳米缓释微粒的抑藻机制，LR型藻毒素（MC-LR）释放风险以及藻细胞类凋亡机制，研究结果对于控制蓝藻水华有重要意义。主要结论如下：. （1）最佳制备条件下，微粒的平均粒径为 1189.5 nm，Zeta 电位为-38.0 mV，包埋率为 52.4%-68%，理论释放天数160天，抑藻活性物质和缓释体外壳通过化学作用结合，具有较好的热稳定性，抑藻效果持续30天内，藻处于非生长状态。在不同介质中的释放机制为骨架的溶蚀控制、扩散和骨架溶蚀协同作用。. （2）纳米缓释颗粒作用于藻细胞的靶基因主要为：PSII反应中心的核心基因psbA和膜修复蛋白基因fabZ在微粒胁迫下表达量分别大幅下调至对照组的63%和32%，说明藻细胞机能损伤一定程度上受基因调控因而损伤过程不可逆。. （3）藻细胞胞、内外MC-LR总量较空白对照组分别下降了43%和26%。同时藻细胞mcyD基因的表达量下调至空白对照组的48%，指示其产毒能力下降。表明纳米缓释微粒不会诱导铜绿微囊藻MC-LR的大量产生及释放，从而降低藻毒素污染风险。. （4）对大型蚤和斑马鱼的慢性毒性、急性毒性（48 h EC50、48 h LC50）及酶活性指标的研究表明，缓释颗粒毒性较低，对高级水生生物更安全。将纳米缓释抑藻颗粒抑藻剂投入实际水样中时，缓释性能良好，抑制率可达81%， MC-LR含量没有增加，说明纳米缓释颗粒抑藻剂具有较好的生态安全性和应用推广前景。