《Aggregate》：Hierarchical Self-Assembled Nanoparticles for Efficient and Eco-Friendly Herbicide Delivery
在现代农业中，化学农药对保障作物产量和粮食安全至关重要。然而，传统农药在施用过程中常因环境因素导致大量损失，利用率低下，并带来生态风险。为应对这一挑战，本研究开发了一种基于层级自组装策略的纳米除草剂递送系统。
1 引言
全球人口增长对粮食生产提出了更高要求，而杂草导致的减产可达20%–50%。尽管已有近2000种除草剂商业化，但其药效常受环境胁迫影响。纳米农药被视为变革性创新，但多数系统依赖材料载体，其实际应用受成本效益和风险评估限制。超分子自组装为构建可调谐、生物相容的纳米农药提供了低成本途径。本研究引入了一种双阶段层级自组装策略，整合主客体识别和静电相互作用，以构建高效、生物相容的纳米颗粒。以全球广泛使用的内吸性除草剂敌草隆（Diuron）为模型，其作用机制主要为抑制光系统II。磺丁基醚-β-环糊精（SBE-β-CD）首先与Diuron形成包合物，随后与带正电的壳聚糖（CSH）通过静电共组装形成纳米颗粒。
2 结果与讨论
2.1 分子识别、主客体自组装与静电自组装
纳米颗粒的制备基于Diuron与SBE-β-CD之间的分子识别和主客体自组装。包合物的形成主要由SBE-β-CD疏水腔内水分子的置换驱动，并伴随主客体分子间的范德华力、疏水效应和氢键作用。1H NMR (D2O) of pure SBE-β-CD (bottom) and an aqueous mixture of SBE-β-CD with equimolar Diuron added (top, filtered). (d) 1H-1H 2D NOESY spectrum (D2O) of an aqueous mixture containing equimolar amounts of SBE-β-CD and Diuron (filtered). (e) UV absorption spectra of Diuron solutions with varying mole fractions of SBE-β-CD. (f) Benesi–Hildebrand plot (1/ΔAvs. 1/[SBE-β-CD]) derived from UV absorption data. (g) Linear correlation between the Diuron absorbance at 248.5 nm and mole fraction of SBE-β-CD. (h) Phase solubility diagram of Diuron as a function of SBE-β-CD concentration. (i–k) Powder X-ray diffraction patterns (i), Fourier-transform infrared spectra (j), and differential scanning calorimetry curves (k) of Diuron, SBE-β-CD, and the Diuron/SBE-β-CD inclusion complex.">
研究表明，包合依赖于空间匹配和热力学稳定性。当将水溶性有限的Diuron加入SBE-β-CD水溶液时，混浊悬浮液变得澄清，表明包合增强了表观溶解度。核磁共振氢谱（1H NMR）和二维核奥弗豪泽效应光谱（2D NOESY）证实了Diuron的芳香端以苯环优先的方向插入SBE-β-CD空腔。紫外吸收光谱和相溶解度图分析表明，包合物具有1:1的化学计量比，结合常数K = 675.49 M-1。粉末X射线衍射（PXRD）、傅里叶变换红外光谱（FTIR）和差示扫描量热法（DSC）表征均证实了包合物的形成。
随后，引入生物可降解的天然聚合物壳聚糖，使其与Diuron/SBE-β-CD包合物进行静电自组装。n = 3; the data are presented as mean ± standard deviation). (c) ?-potential of Diuron/SBE-β-CD@CSH nanoparticles at varying mass ratios (independent experiments: n= 3; the data are presented as mean ± standard deviation). (d) Stability evaluation of particle size and ?-potential for Diuron/SBE-β-CD@CSH nanoparticles (mass ratio of 1.8:1) over 16 days at 25°C (independent experiments: n= 3; the data are presented as mean ± standard deviation). (e–g) Particle size distribution (e), SEM (f), TEM images, and corresponding SAED pattern (g) of Diuron/SBE-β-CD@CSH nanoparticles (mass ratio of 1.8:1).">
通过优化质量比，当Diuron/SBE-β-CD与CSH的质量比为1.8:1时，获得了平均粒径为172.63 nm、多分散指数（PDI）为0.09177、ζ电位为25.2 mV的纳米颗粒。高效液相色谱（HPLC）定量显示其载药量（LC）为10.3871%，封装效率（EE）为57.4386%。扫描电子显微镜（SEM）和透射电子显微镜（TEM）图像显示纳米颗粒呈相对均匀的球形，选区电子衍射（SAED）图谱证实其为非晶态。胶体稳定性测试表明，纳米颗粒在最初4天内粒径和ζ电位保持稳定。
2.2 刺激响应释放与光稳定性
研究考察了温度和pH对Diuron/SBE-β-CD@CSH纳米递送系统释放行为的影响。释放曲线显示明显的温度依赖性，72小时后，35°C下的累积释放量达到90.86%，显著高于25°C（82.48%）和15°C（77.49%）。本征溶出速率（IDR）分析也证实了高温下释放增强。n = 3; the data are presented as mean ± standard deviation. (c, d) Intrinsic dissolution rate (IDR) curves of Diuron/SBE-β-CD@CSH at varying temperatures (c) and pH levels (d). Independent experiments: n= 3; the data are presented as mean ± standard deviation. (e, f) First-order kinetic fitting curves for Diuron/SBE-β-CD@CSH release under different temperatures (e) and pH conditions (f). (g) Cumulative residual percentages of active components in Diuron WP and Diuron/SBE-β-CD@CSH under UV irradiation over time. Independent experiments: n= 3; the data are presented as mean ± standard deviation. (h) Pseudo-first-order kinetic degradation curves of Diuron WP and Diuron/SBE-β-CD@CSH nanoparticles under UV irradiation. (i) Schematic illustration showing enhanced photostability of Diuron encapsulated within Diuron/SBE-β-CD@CSH nanoparticles.">
在pH响应方面，酸性条件（pH 4.5）下Diuron释放增强，72小时累积释放达91.96%。动力学建模表明，一级动力学模型能最好地拟合释放数据，而Ritger-Peppas模型得到的释放指数n值在0.45–0.85之间，表明释放机制为非菲克（ anomalous ）传输，即扩散和基质溶胀/松弛共同控制。
在光稳定性方面，经12小时模拟紫外线辐射后，Diuron/SBE-β-CD@CSH保留了80.00%的有效成分，而商业Diuron WP仅保留68.70%。纳米制剂的光降解半衰期延长至33.70小时，比WP的18.56小时提高了81.58%。这种增强的光稳定性归因于SBE-β-CD空腔对Diuron的封装保护以及壳聚糖组分对紫外线的吸收和散射。
2.3 在杂草叶片上的界面性能
优化的润湿性和抗雨性有助于药液在叶面的均匀铺展和吸收。研究表明，Diuron/SBE-β-CD@CSH的表面张力（62.103 mN/m）低于商业WP（68.156 mN/m）和纯水（71.903 mN/m），表明润湿性增强。在小藜叶片上的动态接触角测量也证实了其铺展能力更优。叶片持液量测试显示，纳米制剂的持液量（4.6461 mg/cm2）显著高于WP（1.3593 mg/cm2）和纯水（0.9316 mg/cm2），表明附着力显著提升。n = 3; the data are presented as mean ± standard deviation. (b) Dynamic contact angle images of droplets for pure water, Diuron WP, and Diuron/SBE-β-CD@CSH solutions on Chenopodium serotinumL. leaves. Independent experiments: n= 3; the data are presented as mean ± standard deviation. (c) Liquid-holding capacity of Chenopodium serotinumL. leaves treated with pure water, Diuron WP, and Diuron/SBE-β-CD@CSH solutions. Independent measurements: n= 3; the data are presented as mean ± standard deviation. (d) CFD simulation maps showing volume fraction and velocity distributions for droplets of pure water, Diuron WP, and Diuron/SBE-β-CD@CSH impacting a hydrophobic surface. (e) Sequential images of droplet impact behavior for pure water, Diuron WP, and Diuron/SBE-β-CD@CSH solutions on Chenopodium serotinumL. leaves. (f) Normalized droplet rebound height (Ht/D0) versus time for pure water, Diuron WP, and Diuron/SBE-β-CD@CSH solutions on Chenopodium serotinumL. leaves. (g) Confocal microscopy images of Chenopodium serotinumL. leaf surfaces treated with Diuron WP and Diuron/SBE-β-CD@CSH, before and after simulated rainfall. (h) Retention of active ingredients on leaf surfaces after rain-wash tests for Diuron WP and Diuron/SBE-β-CD@CSH. Independent measurements: n= 3; the data are presented as mean ± standard deviation.">
通过计算流体动力学（CFD）模拟和实验验证了液滴在疏水叶面上的冲击行为。Diuron/SBE-β-CD@CSH液滴表现出延迟的回缩并始终保持附着，其归一化回弹高度最低。抗雨水冲刷测试表明，商业Diuron WP沉积为不规则团块，易被雨水冲走，而Diuron/SBE-β-CD@CSH则形成均匀的纳米颗粒层，嵌在叶片微缝隙中，冲洗后保留率高。共聚焦激光扫描显微镜（CLSM）观察和HPLC定量证实，纳米制剂在叶片上的活性成分保留率（47.90%）显著高于WP（27.96%）。
2.4 吸收、传导与致死性
Diuron/SBE-β-CD@CSH纳米颗粒的平均粒径为172.63 nm，利用SBE-β-CD和壳聚糖的植物相容性来增强吸收和系统分布。CLSM图像显示，无论是根部处理还是叶面处理，纳米颗粒均能在小藜体内有效长距离转运。根部处理后，荧光信号从根表皮经皮层、中柱向上传输至茎和叶脉；叶面处理后，纳米颗粒高效进入细胞内，并与叶绿体共定位，同时能向下通过韧皮部向根部转运。Chenopodium serotinum L. treated with FITC-loaded Diuron/SBE-β-CD@CSH nanoparticles, illustrating nanoparticle distribution (green fluorescence) in roots, stems, and leaves, along with chloroplast autofluorescence (red). (b–e) Visual assessments (b), plant高度 (d), and fresh weights (e) of Chenopodium serotinumL. grown for 20 days, followed by 20-day treatments with pure water, Diuron WP, and Diuron/SBE-β-CD@CSH at various doses. Number of measurements: n= 12, across three independent experiments. (c–g) Visual assessments (c), plant heights (f), and fresh weights (g) of Chenopodium serotinumL. grown for 40 days, followed by similar 20-day treatments. Number of measurements: n= 12 across three independent experiments; the data are presented as mean ± standard deviation. (h, i) Field photographs (h) and fresh weight comparisons (i) of Abutilon theophrastiMedicus (5–6 leaf stage) and Setaria faberiR. A. W. Herrmann (20–30 cm height) 21 days after treatment with Diuron WP and Diuron/SBE-β-CD@CSH. Number of measurements: n= 10, across three independent experiments; the data are presented as mean ± standard deviation.">
温室药效试验表明，在不同生长阶段（20天和40天），Diuron/SBE-β-CD@CSH对小藜的抑制效果均优于商业WP。在4 kg/ha剂量下，纳米制剂对20天苗龄植株的株高和鲜重抑制率分别为36.81%和77.60%，高于WP的33.81%和69.10%。在40天苗龄植株上，纳米制剂在低剂量（1 kg/ha）下仍表现出显著优势。
田间试验在三个玉米田进行，主要杂草为苘麻（5-6叶期）和狗尾草（20-30 cm高）。在1500 g/ha剂量下，纳米制剂表现出更优异的控草效果。处理后21天，对苘麻的防效达到70.09%，对狗尾草的防效为52.93%，而商业WP的防效分别为53.72%和39.09%。此外，对成熟藜（50-60 cm）也观察到了更强的除草活性。
2.5 淋溶性能与环境影响
通过土柱实验模拟降雨，评估了制剂的迁移行为。Diuron/SBE-β-CD@CSH在土壤中的滞留能力显著增强。其最大淋溶出现时间晚于WP，淋溶量更低（第7天为6.36%，WP为9.18%），13天累积淋溶量减少了18.61%。残留分析也证实纳米制剂组有38.24%的Diuron保留在土壤中，高于WP组的22.09%。这种优异的滞留性归因于纳米结构的巨大比表面积以及壳聚糖外壳的正电荷与带负电土壤颗粒间的静电结合。n = 3; the data are presented as mean ± standard deviation. (d, e) Germination status (d) and fresh weights (e) of cucumber seeds after 1 week of treatment with various concentrations of Diuron TC, Diuron WP, and Diuron/SBE-β-CD@CSH. Number of measurements: n= 10 across three independent experiments; the data are presented as mean ± standard deviation. (f, g) Germination status (f) and fresh weights (g) of rice seeds treated similarly for 1 week. Number of measurements: n= 10 across three independent experiments; the data are presented as mean ± standard deviation. (h, i) Mortality rates of zebrafish after exposure to varying concentrations of Diuron TC, Diuron WP, and Diuron/SBE-β-CD@CSH for 72 h (h) and 96 h (i). Number of measurements: n= 10 across three independent experiments; the data are presented as mean ± standard deviation. (j) Survival rates of earthworms after 48 h of treatment with different concentrations of Diuron TC, Diuron WP, and Diuron/SBE-β-CD@CSH. Number of measurements: n= 10 across three independent experiments; the data are presented as mean ± standard deviation.">
对非靶标生物的生态毒理评估显示，Diuron/SBE-β-CD@CSH的安全性更高。在种子发芽试验中，在20 mg/L浓度下，纳米制剂处理的黄瓜和水稻种子总生物量高于原药（TC）和WP处理组。在高浓度下，纳米制剂仍能保持部分发芽率，而TC和WP则完全或严重抑制发芽。对斑马鱼的急性毒性试验表明，纳米制剂在各个时间点的半数致死浓度（LC50）均最高，毒性最低。例如，96小时LC50，WP仅为3.33 mg/L，而纳米制剂约为其4倍。对蚯蚓的急性毒性试验也显示，纳米制剂的LC50（2978.03 mg/L）远高于WP（742.27 mg/L）和TC（1458.45 mg/L）。
2.6 体外细胞毒性与ROS生成
研究评估了Diuron制剂对心肌细胞（AC16）和肝细胞（MIHA）的毒性。细胞活力呈剂量依赖性下降，Diuron WP的细胞毒性最强。在96 mg/L浓度下，WP处理的MIHA和AC16细胞活力分别降至56%和12%以下，而纳米制剂组则保持在83%和43%。活/死细胞染色也显示，纳米制剂处理组的死细胞比例显著减少。活性氧（ROS）检测表明，WP处理组细胞内ROS水平升高至对照组的2.5倍以上，而纳米制剂组则表现出最显著的抗氧化效应，ROS生成最少。这可能归因于纳米封装减少了细胞对Diuron的直接暴露，以及壳聚糖的羟基和氨基具有清除自由基的能力。
2.7 大鼠体内安全性评价
通过大鼠32天每日灌胃给药进行体内毒性评估。与对照组和纳米制剂组相比，Diuron TC和WP组大鼠体重增加较少。肝毒性指标血清丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）在TC和WP组显著升高，其中WP组最高。纳米制剂处理则使ALT和AST降低了约20%，保持在正常参考范围附近。心脏毒性指标肌酸激酶（CK）和乳酸脱氢酶（LDH）在WP组急剧升高，约为对照组的2倍和1.74倍，超过了正常范围。纳米制剂显著减轻了这种损害，使CK和LDH降低了约28%。肾脏毒性指标变化不大。血液学分析显示，纳米制剂组的白细胞（WBC）计数升高幅度最小，表明炎症反应和系统毒性减弱。n = 5 across three independent experiments; the data are presented as mean ± standard deviation. (b) Quantitative analysis of liver function biomarkers (AST and ALT) in rat blood after 32 days of daily oral administration of Diuron TC, Diuron WP, and Diuron/SBE-β-CD@CSH. Number of measurements: n= 5 across three independent experiments; the data are presented as mean ± standard deviation. (c) Quantitative analysis of myocardial function biomarkers (CK and LDH). Number of measurements: n= 5 across three independent experiments; the data are presented as mean ± standard deviation. (d) Quantitative analysis of kidney function biomarkers (BUN and CREA). Number of measurements: n= 5 across three independent experiments; the data are presented as mean ± standard deviation. (e) Quantitative analysis of RBC, WBC, and PLT. Number of measurements: n= 5 across three independent experiments; the data are presented as mean ± standard deviation. (f) H&E staining images of rat heart, liver, spleen, lung, and kidney tissues following 32-day treatment. (g) Fluorescence images of 4-HNE staining in
