Graphene oxide activates canonical TGFß signalling in a human chondrocyte cell line via increased plasma membrane tension.

Graphene Oxide (GO) has been shown to increase the expression of key cartilage genes and matrix components within 3D scaffolds. Understanding the mechanisms behind the chondroinductive ability of GO is critical for developing articular cartilage regeneration therapies but remains poorly understood. The objectives of this work were to elucidate the effects of GO on the key chondrogenic signalling pathway - TGFß and identify the mechanism through which signal activation is achieved in human chondrocytes. Activation of canonical signalling was validated through GO-induced SMAD-2 phosphorylation and upregulation of known TGFß response genes, while the use of a TGFß signalling reporter assay allowed us to identify the onset of GO-induced signal activation which has not been previously reported. Importantly, we investigate the cell-material interactions and molecular mechanisms behind these effects, establishing a novel link between GO, the plasma membrane and intracellular signalling. By leveraging fluorescent lifetime imaging (FLIM) and a membrane tension probe, we reveal GO-mediated increases in plasma membrane tension, in real-time for the first time. Furthermore, we report the activation of mechanosensory pathways which are known to be regulated by changes in plasma membrane tension and reveal the activation of endogenous latent TGFß in the presence of GO, providing a mechanism for signal activation. The data presented here are critical to understanding the chondroinductive properties of GO and are important for the implementation of GO in regenerative medicine.

Defect-free graphene enhances enzyme delivery to fibroblasts derived from patients with lysosomal storage disorders.

Enzyme replacement therapy shows remarkable clinical improvement in treating lysosomal storage disorders. However, this therapeutic approach is hampered by limitations in the delivery of the enzyme to cells and tissues. Therefore, there is an urgent, unmet clinical need to develop new strategies to enhance the enzyme delivery to diseased cells. Graphene-based materials, due to their dimensionality and favourable pattern of interaction with cells, represent a promising platform for the loading and delivery of therapeutic cargo. Herein, the potential use of graphene-based materials, including defect-free graphene with positive or negative surface charge and graphene oxide with different lateral dimensions, was investigated for the delivery of lysosomal enzymes in fibroblasts derived from patients with Mucopolysaccharidosis VI and Pompe disease. We report excellent biocompatibility of all graphene-based materials up to a concentration of 100 µg mL-1 in the cell lines studied. In addition, a noticeable difference in the uptake profile of the materials was observed. Neither type of graphene oxide was taken up by the cells to a significant extent. In contrast, the two types of graphene were efficiently taken up, localizing in the lysosomes. Furthermore, we demonstrate that cationic graphene flakes can be used as carriers for arylsulfatase B enzyme, for the delivery of the lacking enzyme to the lysosomes of Mucopolysaccharidosis VI fibroblasts. Arylsulfatase B complexed with cationic graphene flakes not only retained the enzymatic activity, but also exerted biological effects almost twice as high as arylsulfatase B alone in the clearance of the substrate in Mucopolysaccharidosis VI fibroblasts. This study lays the groundwork for the potential use of graphene-based materials as carriers for enzyme replacement therapy in lysosomal storage disorders.