纳米流式细胞仪检测EV和LNP的检测实践

doi:10.5246/jcps.2025.10.070

摘要/Abstract

摘要：

脂质纳米颗粒(LNPs)和细胞外囊泡(EVs)是新兴的治疗平台, 具有独特的优势。LNPs由四种关键脂质组成, 可有效地递送各种药物或遗传物质, 如FDA批准的Onpattro和COVID-19 mRNA疫苗。由细胞天然产生的EV可以高效靶向递送生物活性分子, 如基于EV的Bexsero疫苗。由于LNP和EV尺寸小、异质性强, 流式细胞术(FCM)等精确分析技术对于LNP和EV表征比常用的NTA(纳米粒子跟踪分析)和DLS(动态光散射)更全面。这项研究利用纳米流式细胞仪来开发纳米药物检测策略, 详细阐述了检测过程, 以确保分析的准确性和重复性。

关键词: 脂质纳米粒, 细胞外囊泡, 纳米流式细胞仪

Abstract:

Lipid nanoparticles (LNPs) and extracellular vesicles (EVs) have emerged as powerful therapeutic platforms, each offering distinct advantages. LNPs, composed of four essential lipid components, enable efficient delivery of drugs and genetic materials, underpinning FDA-approved therapies such as Onpattro and the COVID-19 mRNA vaccines. EVs, naturally secreted by cells, transport bioactive molecules with remarkable delivery efficiency and reduced off-target effects, as exemplified by the EV-based Bexsero vaccine. Given their small size and intrinsic heterogeneity, precise characterization is critical. Compared to conventional techniques like nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS), flow cytometry (FCM) offers a more comprehensive analytical profile for both LNPs and EVs. In the present study, we leveraged a nano-FCM to advance detection strategies for nanoscale therapeutics. A detailed workflow is presented to ensure analytical precision and reproducibility.

Key words: Lipid nanoparticles, Extracellular vesicles, Nano-flow cytometer

Supporting:

孙玉芳, 许迎利, 于化龙, 宋书香. 纳米流式细胞仪检测EV和LNP的检测实践[J]. 中国药学(英文版), 2025, 34(10): 943-953.

Yufang Sun, Yingli Xu, Hualong Yu, Shuxiang Song. Application of nano-flow cytometry for the detection of extracellular vesicles and lipid nanoparticles[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(10): 943-953.

图/表 6

Figure 1. (a) Threshold and flow rate check; (b) Scatter check; (c) Fluorescence check; (d) Sensitivity monitoring.

Figure 2. (a) Noise of the instrument; (b) The 5-nm filter of the cytometer; (c) PBS without or with 20-nm filtration; (d) The cytometer built-in cleaning and inspection program; (e) Different tubes with different backgrounds.

Figure 3. (a) Linearity of sample dilution; (b) High concentration with swarm; (c) Proper concentration without swarm; (d) Dye with aggregation.

Figure 4. (a) Gate the sample particles; (b) Gate the single sample particles; (c) Gate the GFP+ EVs.

Figure 5. (a) Gate the sample particles; (b) Gate the single sample particles; (c) Gate the LNP with mRNA encapsulation.

Figure 6. (a) Half-angle; (b) Collected median vs scattering cross-section; (c) Model fit by comparing the collected median data for each bead size; (d) Instrument’s scattering performance profile in relation to the RI; (e) Size distribution of EVs; (d) Size distribution of LNPs.

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