Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven 5612AZ, The Netherlands.
Department of Applied Physics, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Den Dolech 2, 5600MB Eindhoven, The Netherlands.
ACS Nano. 2022 Mar 22;16(3):3785-3796. doi: 10.1021/acsnano.1c08277. Epub 2022 Mar 11.
Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.
抗体功能化纳米颗粒 (NPs) 常用于提高对目标细胞的靶向选择性。在分子水平上，NP 表面上的功能抗体数量和靶细胞上受体的密度决定了靶向相互作用。为了合理开发选择性 NPs，非常希望能够对这两个参数进行单分子定量。然而，能够以纳米分辨率计数分子的技术却很少。在这里，我们开发了一种标记方法，使用直接随机光学重建显微镜 (dSTORM) 来定量测定与 NPs 共轭的功能西妥昔单抗的数量和乳腺癌细胞中表皮生长因子受体 (EGFRs) 的表达。dSTORM 的单分子分辨率允许在纳米尺度上定量分子，深入了解单个 NP 配体和细胞受体的分布。此外，我们使用几何模型预测了可及抗体缀合 NPs 的分数，表明总数量超过了可及抗体数量。最后，我们将 NP 功能、细胞受体密度和 NP 摄取相关联，以确定具有最高细胞摄取选择性的区域。我们的结论是，使用 dSTORM 进行单分子功能作图提供了对 NP 靶向的分子理解，有助于合理设计选择性纳米药物。
