Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege út 29-33, BudapestH-1120, Hungary.
Department of Biological Physics, Eötvös University, BudapestH 1117, Hungary.
ACS Appl Bio Mater. 2023 Jan 16;6(1):64-73. doi: 10.1021/acsabm.2c00595. Epub 2022 Oct 14.
Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [ = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.
功能化纳米颗粒（NPs）由于能够穿透活细胞，因此被广泛应用于靶向药物输送和生物医学成像。大多数细胞的外层是富含糖的细胞糖萼层，该层可能在任何摄取过程中都起着重要作用。然而，细胞糖萼在 NP 摄取中的确切作用仍未被揭示。在这里，我们原位监测了带正电荷的碱性硫醇（TMA）功能化的金 NPs 进入附着癌细胞的细胞内摄取情况，其中一些细胞预先用糖萼消化酶进行了糖萼消化。糖萼的蛋白聚糖（PG）成分用软骨素 ABC 酶处理。该酶作用于硫酸软骨素 4-硫酸盐、硫酸软骨素 6-硫酸盐和硫酸皮肤素，对透明质酸盐的作用较慢。HeLa 细胞的摄取测量是通过应用基于谐振波导光栅的高通量无标记光学生物传感器来完成的。带正电荷的金 NPs 分别使用不同的尺寸[=2.6、4.2 和 7.0nm，分别为小（S）、中（M）和大（L）]。还在对照实验中使用了带负电荷的柠檬酸根封端的单宁酸（CTA，=5.5nm） NPs。实时生物传感器数据证实了功能化 NPs 的细胞摄取，这通过透射电子显微镜得到了直观的证实。研究发现，酶消化促进了带正电荷的 S-和 M-尺寸 NPs 的进入，M-尺寸的 NP 进入效果更为明显。还使用了其他消化糖萼不同成分的酶，并对结果进行了比较。消化糖胺聚糖的肝素酶 III 处理也增加了 NP 的摄取，而消化糖蛋白和糖脂的神经氨酸酶则降低了 HeLa 细胞对 NP 的摄取。这表明，带正电荷的 NPs 的摄取增加是由于细胞糖萼的糖蛋白和糖脂成分增加，而硫酸乙酰肝素减少所致。我们的研究结果提出了一个假设，即 2-4nm 带正电荷的 NPs 的细胞摄取是由糖萼的糖蛋白和糖脂成分促进的，但被 PG 抑制。
