Multimodal nanoparticles (gold in polylactide) and their effect on three-dimensional cell cultures as organ models

我们将合成多模态纳米颗粒，可以在细胞中被跟踪，并通过特定的荧光标记在内切溶酶体中被检测到。这将通过将荧光标记的生物分子共价结合到小的金纳米颗粒(直径约。10nm)，它们的荧光被淬灭。这些将被纳入聚乳酸-共聚物（PLGA）颗粒中（估计：大约10个金纳米颗粒在一个直径约100纳米的PLGA颗粒中）。PLGA包膜也将与荧光染料共价功能化。多模态纳米颗粒的细胞摄取将通过原位共聚焦激光扫描显微镜使用3D细胞培养模型（隐窝类器官和肿瘤球体）进行定量研究。我们将重点关注纳米颗粒在3D细胞培养或类器官的复杂细胞网络中的途径，并研究多模态纳米颗粒和生物分子的影响。用于基因沉默的生物分子（siRNA）将被偶联到金纳米颗粒上以实现生物效应。首先，将研究荧光eGFP作为模型蛋白的基因沉默，然后研究重要炎症疾病介质的基因沉默，如TNF-α、KC、IP 10和抑制凋亡的survivin作为额外的蛋白质模型。纳米颗粒介导的基因沉默将证明并扩展我们的概念到另一个治疗相关的靶标。总之，我们的目标是更好地了解功能性纳米颗粒的生物学效应，包括剂量、途径和命运等问题。三维细胞培养模型应作为“经典”二维细胞培养和动物模型之间的桥梁。我们还希望实现二维和三维细胞培养模型的定量验证与生物活性纳米颗粒。

We will synthesise multimodal nanoparticles that can be tracked in the cell and detected in the endo-lysosome by specific fluorescence labels. This will be achieved by covalently binding fluorescently labelled biomolecules to small gold nanoparticles (diameter approx. 10 nm) where their fluorescence is quenched. These will be incorporated into polylactide-co-glycolide (PLGA) particles (estimation: approximately 10 gold nanoparticles within a single PLGA particle with a diameter of around 100 nm). The PLGA envelope will also be covalently functionalised with fluorescent dyes. The cellular uptake of the multimodal nanoparticles will be studied quantitatively using 3D cell culture models (crypt organoids and tumour spheroids) by in situ confocal laser scanning microscopy. We will focus on the pathway of the nanoparticles inside the complex cell network of the 3D cell culture or organoids and investigate the impact of the multimodal nanoparticles and biomolecules. Biomolecules for gene silencing (siRNA) will be conjugated to the gold nanoparticles to achieve a biological effect. First, the gene silencing of fluorescent eGFP as model protein will be investigated, followed by the gene silencing of important inflammatory disease mediators, such as TNF-α, KC, IP 10, and the apoptosis-inhibiting survivin as an additional protein model. The nanoparticle-mediated gene silencing of survivin will prove and extend our concept to another therapeutically relevant target.In summary, we aim for a better understanding of the biological effects of functional nanoparticles, covering inter alia the questions of dose, pathway, and fate. Three-dimensional cell culture models shall serve as bridge between "classical" two-dimensional cell cultures and animal models. We also want to achieve a quantitative validation of two-dimensional and three-dimensional cell culture models with respect to biologically active nanoparticles.