Burkitt Sean, Eremina Olga E, Mouchawar Anthony, Fernando Augusta, Edmondson Elijah, Stern Stephan, Marks Carolyn, Zavaleta Cristina
Department of Biomedical Engineering, University of Southern California, 3650 McClintock Avenue, Los Angeles, California 90089, United States.
Michelson Center for Convergent Bioscience, University of Southern California, 1002 Childs Way, Los Angeles, California 90089, United States.
ACS Nano. 2025 Aug 19;19(32):29286-29300. doi: 10.1021/acsnano.5c05069. Epub 2025 Aug 4.
Gold nanoparticles (AuNPs) are increasingly used in applications across the biomedical domain, yet their long-term biodistribution and biocompatibility remain poorly understood. Conventional brightfield microscopy imaging techniques often fail to detect AuNPs due to optical diffraction limits and lack of chromogenic contrast. Understanding the biodistribution and ultimate fate of these nonbiodegradable NPs is crucial for further development of AuNP-based therapeutics and diagnostics. Here, we present a label-free multiphoton luminescence (MPL) imaging workflow that enables sensitive detection of AuNPs in liver histology sections, even 1 year after intravenous (IV) administration. MPL imaging exploits the intrinsic nonlinear optical properties of AuNPs to generate broadband emission under ultrafast pulsed laser excitation, enabling subcellular localization without exogenous labels while having the ability to rapidly image entire organ sections. The intrinsic, distinct broadband MPL emission produced by gold allows us to study these NPs in their biological context without extrinsic labels while also faithfully representing the surrounding tissue architecture via autofluorescence and second harmonic generation. We demonstrate that MPL imaging detects up to 98% more AuNP-positive regions than brightfield microscopy in challenging low-dose (1 nM) conditions and requires no modification of standard histology workflows. Correlative imaging with SEM-EDS confirms high spatial specificity (AUC = 0.955) of MPL for AuNP localization. Dose-dependent retention patterns were observed across liver tissue, and MPL analysis showed strong correlation with ICP-MS quantification. Importantly, histological and immunohistochemical analyses (Masson's trichrome, CD3, and TUNEL) revealed no significant fibrosis, immune activation, or apoptosis in liver tissue at either low (1 nM) or high (10 nM) doses at 1 year post IV administration. These findings establish MPL imaging as a robust, label-free tool for long-term tracking of AuNPs in biological tissue and highlight its potential for improving biodistribution and safety assessments.
金纳米颗粒（AuNPs）在生物医学领域的应用越来越广泛，但其长期生物分布和生物相容性仍知之甚少。由于光学衍射极限和缺乏发色对比度，传统的明场显微镜成像技术常常无法检测到AuNPs。了解这些不可生物降解纳米颗粒的生物分布和最终归宿对于基于AuNP的治疗和诊断的进一步发展至关重要。在此，我们展示了一种无标记多光子发光（MPL）成像工作流程，即使在静脉注射（IV）一年后，也能在肝脏组织切片中灵敏地检测到AuNPs。MPL成像利用AuNPs的固有非线性光学特性，在超快脉冲激光激发下产生宽带发射，无需外源性标记即可实现亚细胞定位，同时能够快速对整个器官切片进行成像。金产生的固有、独特的宽带MPL发射使我们能够在不使用外源性标记的情况下，在其生物学背景中研究这些纳米颗粒，同时还能通过自发荧光和二次谐波产生忠实地呈现周围的组织结构。我们证明，在具有挑战性的低剂量（1 nM）条件下，MPL成像检测到的AuNP阳性区域比明场显微镜多98%，并且无需修改标准组织学工作流程。与扫描电子显微镜-能谱仪（SEM-EDS）的相关成像证实了MPL对AuNP定位具有高空间特异性（AUC = 0.955）。在肝脏组织中观察到剂量依赖性保留模式，MPL分析显示与电感耦合等离子体质谱（ICP-MS）定量有很强的相关性。重要的是，组织学和免疫组织化学分析（Masson三色染色、CD3和TUNEL）显示，静脉注射一年后，低剂量（1 nM）或高剂量（10 nM）的肝脏组织中均未出现明显的纤维化、免疫激活或细胞凋亡。这些发现确立了MPL成像作为一种强大的、无标记的工具，用于在生物组织中对AuNPs进行长期追踪，并突出了其在改善生物分布和安全性评估方面的潜力。
