单颗粒水平脂质纳米药物多参数定量表征技术的建立

Nanomedicine, particularly lipid-based nanomedicine, holds great potential in revolutionizing disease diagnosis and treatment. However, nanomedicine development is held up by the lack of nanoparticle characterization. The intrinsic heterogeneity of nanomedicine calls for the development of high-throughput technique that can analyze nanoparticles on a single-particle basis. Taking advantage of the high sensitivity, rapid speed, and multiparameter and quantitative analysis capability of the high sensitivity flow cytometry (HSFCM) developed in our laboratory, this project is intend to build up a single-particle platform for the comprehensive characterization of lipid-based nanomedicine. This is achieved by simultaneous measurement of the light scatter and multiple fluorescence signals emitted from single nanomedicine particles as they transit individually through the highly focused laser beam. This platform will provide effective and practical characterization methods for the synthesis optimization and quantity control of lipid-based nanomedicine. We will prepare doxorubicin-encapsulated liposome with monoclonal antibody conjugated on the surface for targeted delivery, and use it as the model system for method development. We plan to build the comprehensive method for lipid-based nanomedicine characterization that can provide all the information including particle size and distribution, particle concentration, drug-encapsulation efficiency, drug-loading content and its distribution, and antibody density and distribution. This newly established method will be applied to study the effects of drug-to-lipid ratio and different drug-loading approaches on drug-loading efficiency. Effects of drug-loading content and antibody density on cytotoxicity of cancer cells will be investigated at high precision. This novel method aims to meet the challenge of nanomedicine characterization and to promote the development of nanomedicine.

纳米药物，尤其是脂质纳米药物，具有重要的生物医学应用前景，然而表征技术的匮乏已严重阻碍纳米医药的发展。纳米药物的高度异质性迫切需要发展高通量的单颗粒表征技术。本项目拟利用实验室创建的超高灵敏流式检测技术灵敏、快速、多参数、定量分析的独特性能，通过对单个脂质纳米颗粒散射光和多色荧光信号的同时检测，建立脂质纳米药物的单颗粒水平综合表征平台，为脂质纳米药物的合成优化、质量控制等提供高效、实用的表征技术和方法。将合成靶向型阿霉素脂质体，并以此为模型建立集颗粒粒径及其分布、颗粒浓度、包封率、载药量及其分布、表面偶联抗体密度及其分布等多种指标于一体的脂质纳米药物综合表征方法；应用于阿霉素梯度载药和载药方式的优化，并在细胞水平考察单克隆抗体密度、阿霉素载药量的药效关系。该项目的顺利开展将突破传统技术仅能对脂质纳米药物的单个参数进行宏观测定的表征困境，有力地推动纳米药物的研发进程。

脂质纳米药物是目前临床上最受认可的载药系统。基于纳米药物固有的高度异质性，需要对其粒径及其分布、载药量及其分布、颗粒浓度、药物包封率、表面偶联靶向配体数量及其分布等物理、化学性状进行快速、准确的单颗粒水平表征，以获得具有统计代表性的数据。我们制备了阿霉素脂质体并以此为模型，利用实验室自行研制的具有国际领先水平的超高灵敏流式检测装置（high sensitivity flow cytometer, HSFCM），通过对单个脂质体散射和荧光信号的同时检测，借助粒径标准球和荧光亮度标准球，建立了脂质纳米药物的粒径及其分布、载药量及其分布、颗粒浓度和载药率的快速、高分辨定量表征方法，检测速率高达10,000颗粒/分钟，实现了对商品化阿霉素脂质体Doxoves和gDoxil粒径和载药量的快速表征。将靶向疾病组织或肿瘤细胞的配体分子（如抗体、核酸适配体、短肽或特异性配体等）偶联在纳米颗粒的表面是实现纳米药物靶向治疗的前提。我们合成了表面偶联叶酸配体的脂质体，采用荧光标记的叶酸受体作为探针测定脂质体表面可利用叶酸的含量，以粒径和荧光当量已知的二氧化硅纳米颗粒作为标准品，实现了脂质体粒径和表面叶酸含量的同时测定，建立了叶酸脂质体表面可利用配体密度及其分布的定量表征方法。采用传统流式细胞仪考察了配体密度对脂质体与细胞结合能力的影响，得到脂质体的最佳配体密度。此外，我们还考察了叶酸脂质体制备方法、PEG长度等参数对脂质体表面可利用配体数量的影响。结合免疫荧光标记，我们将该方法拓展至以转铁蛋白和Her2单克隆抗体分别作为靶向配体的脂质体表面配体含量的表征，并建立了一种表征单个脂质体表面总蛋白含量及可利用配体含量的表征方法，可测定脂质体表面有效配体的比例。最后，我们制备了以转铁蛋白为靶向基元、载有阿霉素的脂质体颗粒，并对HSFCM进行双激光升级，建立了对脂质体纳米药物的粒径、载药量和表面配体含量进行多参数同时表征的新方法，将为靶向纳米药物的合成优化、质量控制及细胞摄入研究等提供先进的测量技术。

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