Three-Dimensional Quantitative Co-Mapping of Pulmonary Morphology and Nanoparticle Distribution with Cellular Resolution in Nondissected Murine Lungs

Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex vivo murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 ± 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 ± 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity–dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines.

由于缺乏有效的成像工具，以细胞分辨率解析完整器官内纳米颗粒（nanoparticles, NPs）的生物分布、生物动力学及生物学效应，目前仍在很大程度上难以实现。本研究验证了结合光学组织透明化（optical tissue clearing）的光片荧光显微镜（light sheet fluorescence microscopy），可在未解剖的离体小鼠肺组织中，以细胞分辨率同时完成肺形态与纳米颗粒生物分布的三维标绘。组织自发荧光可实现整个支气管树、腺泡结构及血管的无标记定量形态计量（label-free, quantitative morphometry）。将荧光标记纳米颗粒与肺形态进行共配准（co-registration）后发现，根据给药途径的不同，肺部纳米颗粒的分布存在显著差异：包括麻醉状态下的气管内滴注（intratracheal instillation）与呼吸机辅助气溶胶吸入（ventilator-assisted aerosol inhalation）两种方式。与气管内滴注组相比，气溶胶吸入组的传导气道（conducting airways）与腺泡内纳米颗粒分布更为均匀：吸入组的中心-外周（C/P）纳米颗粒沉积比为1（0.98±0.13），而滴注组的中央沉积量较外周提升2倍，其C/P比为1.98±0.37。正如计算流体动力学（computational fluid dynamics）模拟所预测的那样，吸入给药后大多数纳米颗粒分布于腺泡的近端区域。在细胞分辨率下，可观察到细支气管与腺泡内存在斑片状的纳米颗粒沉积，且气管内滴注组的沉积现象更为显著。荧光强度-剂量响应曲线（fluorescence intensity–dose response curve）具有极佳的线性度，可实现精准的纳米颗粒剂量测定，并揭示约5%的吸入气溶胶会沉积于肺部。这一单模态成像技术可实现组织架构与纳米颗粒生物分布的定量共配准，有望加速阐明完整组织内纳米颗粒的生物动力学与生物活性，从而助力纳米毒理学研究与纳米药物（nanomedicines）的开发。