摘要目的:构建新型冠状病毒高亲和力广谱双价纳米抗体A2-A2，了解气雾化后的稳定性及给药效率。方法:原核表达和亲和纯化获得A2-A2。采用假病毒中和实验和生物膜层干涉实验评价A2-A2的中和能力和亲和力。肺部液体雾化给药器对A2-A2进行雾化，利用Western印迹法、ELISA、纳米颗粒跟踪分析技术评价A2-A2雾化前后性能和稳定性，同时通过雾化给药和腹腔注射2种方式对小鼠进行活体成像荧光分析和各器官抗体浓度检测，评估雾化式给药A2-A2在小鼠体内存留情况和递送到肺部的效率。结果:成功构建了新型冠状病毒双价纳米抗体A2-A2。A2-A2与SARS-CoV-WT、Alpha、Beta、Gamma、Delta、Omicron B.1.1.529、Omicron BA.5等毒株均具有良好的广谱中和作用和亲和力，中和活性的半数抑制浓度最低为0.3 μg/mL，亲和力最低为10 -12。雾化前后A2-A2活性、结构及颗粒大小（100~150 nm粒径范围占比71.3%）均未发生显著改变，具有良好的稳定性。A2-A2雾化式给药在小鼠肺部活体成像中留存近5 h，在小鼠解剖后肺中留存近24 h，浓度可达1.2 mg/mL，显著优于腹腔注射给药方式。 结论:双价纳米抗体A2-A2具有良好的稳定性，可通过雾化给药的方式有效到达肺部。相比于腹腔注射方式，A2-A2在肺部存留时间较长且具有更高的浓度。

abstractsObjective:To construct a SARS-CoV-2 high-affinitive broad-spectrum bivalent nanobody A2-A2, and evaluate its stability and delivery efficiency after aerosolization.Methods:A2-A2 was obtained through prokaryotic expression and affinity purification. The neutralizing ability and affinity of A2-A2 were assessed using pseudovirus neutralization assays and biolayer interferometry. A2-A2 was aerosolized using a pulmonary liquid atomizer, and its properties and stability before and after aerosolization were evaluated by Western blotting, ELISA, and nanoparticle tracking analysis. In vivo imaging fluorescence analysis and antibody concentration assays in various organs of mice were conducted using both aerosolized administration and intraperitoneal injection of A2-A2, so as to evaluate the retention and delivery efficiency of aerosolized A2-A2 in the lungs. Results:The SARS-CoV-2 bivalent nanobody A2-A2 was successfully constructed. A2-A2 exhibited broad-spectrum neutralizing activity and affinity against SARS-CoV-WT, Alpha, Beta, Gamma, Delta, Omicron B.1.1.529 and Omicron BA.5, with the lowest half-maximal inhibitory concentration of 0.3 μg/mL and an affinity of 10 -12. No significant changes in activity, structure, or particle size of A2-A2 (with the particle size range of 100-150 nm accounting for 71.3%) were observed before and after aerosolization, indicating good stability. Aerosolized A2-A2 was retained in the lungs of mice for nearly 5 hours in vivo, and persisted in the lungs of dissected mice for nearly 24 hours with a concentration of up to 1.2 mg/mL, significantly outperforming intraperitoneal injection. Conclusions:The bivalent nanobody A2-A2 exhibits good stability and can be effectively delivered to the lungs via aerosolization. Compared with intraperitoneal injection, aerosolized A2-A2 has a longer retention time and higher concentration in the lungs.